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Popliteal lymph node enlargement induced in syngeneic hosts by T cells from foster-nursed mice
CELLULAR
IMMUNOLOGY
128, 175%184(1990)
Popliteal Lymph Node Enlargement Induced in Syngeneic Hosts by T Cells from Foster-Nursed Mice IRENENEPOMNASCHY,‘ADRIANAD~ROCHE,’SANDRATORELLO,’ ALEJANDRAGOLDMAN,VALERIA BUGGIANO,* CHRISTIANEDOSNEPASQUALINI,’ ANDISABELPIAZZON’ Divisicin Medicina Experimental, Instituto de Investigaciones Hematol6gicas. Academia National de Medicina, Las Heras 3092, 1425 Buenos Aires, Argentina Received October 5. 1989; acceptedJanuary 13, 1990 Splenocytes from adult Fl mice were assayed for their capacity to induce popliteal lymph node enlargement (PLNE) when inoculated in the footpad of identical or reciprocal FI hosts. The results obtained showed that: (i) T Lyt l+ splenocytes from adult Fl hybrids were able to induce a significant PLNE when inoculated in reciprocal but not in identical Fl hosts.(ii) Fosternursing of Fl hybrids on mothers from the paternal strain was able to induce permanent alterations in the ability of their T splenocytes to induce PLNE: Lyt l+ splenocytes were able to induce significant PLNE in identical but not in reciprocal Fl hosts.Thus, the ability of T splenocytes from foster-nursed Fl hybrids to induce PLNE resembled that observed in reciprocal Fl hybrids nursed by their own mothers. (iii) PLNE was accompanied by cell proliferation involving host B and T lymphocytes. (iv) This PLNE could be detected using Fl hybrids from parental strains differing or not at H-2 antigens but involving a parental strain expressing the stimulatory MIS” allele and a parental strain bearing the nonstimulatory Mlsb allele while it was not observed in F 1 hybrids from parental strains sharing MIS” antigens. o 1990 Academic PXSS, hc.
INTRODUCTION Matemofetal relationship involves not only pregnancy but also breast feeding. Colostrum and milk are rich in immunoglobulins, macrophages, and T and B lymphocytes (1). Weiler et al. (2) have reported that milk cells may penetrate the stomach wall and colonize the neonatal rodents. It has been proposed that the exposure of the neonate to maternal antigens during breast-feeding may alter the immunologic reactivity of the litter toward maternal allografts (3). In a previous paper (4) we have reported that splenocytes from reciprocal Fl hybrids differ in their ability to stimulate T cell proliferation in syngeneic mixed lymphocyte reactions. Foster-nursing of Fl hybrids on mothers from the paternal strain was able to induce permanent alterations in the ability of their splenocytes to stimulate T cell proliferation in these reactions. The stimulatory ability of splenocytes from r Member of the Research Career of the Consejo National de Investigaciones Cientificas y Tknicas, CONICET. * Fellowship of CONICET. 175 0008-8749/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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NEPOMNASCHY
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foster-nursed hybrids became indistinguishable from that observed in the reciprocal Fl combination nursed by its own mother. Localized proliferative in vivo responsesto subcutaneously injected syngeneic cells have previously been described. Popliteal lymph node enlargement (PLNE) has been induced in normal mice by inoculating syngeneic T cells from aged mice (5), and neonatally (6) or adult (7) thymectomized mice. L’Age-Stehr and Diamantstein have reported (8) that splenocytes from cyclophosphamide-pretreated mice were also able to induce PLNE when inoculated in normal syngeneic mice. In this paper, we show that lactation is responsible for the ability of Lyt 1+ T splenocytes from Fl hybrids to induce a significant PLNE when inoculated in reciprocal Fl hosts. MATERIALS AND METHODS Mice The following pairs of reciprocal Fl hybrids, raised in our colony were used: (BALB/cXAKR)Fl (H-2dXH-2k) (MLSbXMlsa) and (AKRXBALB/c)Fl; (BALB/ cXAKR.M/Sn)Fl (H-2dXH-2m) (MlsbXMlsa) and (AKR.M/SnXBALB/c)Fl; (BALB/cXDBA/2)Fl (H-2dXH-2d) (MlsbXMls”) and (DBA/2XBALB/c)Fl; (AKRXDBA/2)Fl (H-2kXH-2d) (Mls”XMls”) and (DBA/2XAKR)Fl; (AISRXAKR.M/ Sn)Fl (H-2kXH-2m) (Mls”XMls”) and (AKR.M/SnXAKR)Fl. Animals from both sexesand 2- to 4-months old were used. The maternal strain of the F 1 hybrid is always denoted first. Foster-Nursing Full-term pregnant females were placed in special cagesto avoid any suckling by the litter; immediately after birth, half of the litter was returned to its own mother and the other half was foster-nursed on mothers from the paternal strain which had been pregnant from syngeneic males. Foster-nursed mice were denoted as (AXB)fBFl. They were used when they were adults (2-4 months) and, in some experiments, when 8- to 12-month old. Cell Suspensions Splenocytes were obtained aseptically by gently pressing the spleen through a stainless-steel mesh. Viability was assessedby trypan blue exclusion (over 90% was required for use) after which the cells were resuspended at the desired concentration in RPM1 1640 (GIBCO, Grand Island, NY)). Spleen populations enriched in T cells (NWNA) were obtained by passagethrough nylon wool columns as described by Julius et al. (9). Antisera Treatment The anti-Thy 1.2., anti-Lyt 1, and anti-Lyt 2 sera were obtained from BectonDickinson Labware, Oxnard; goat anti-mouse immunoglobulins (IgE+IgG+IgM) (H&L) (anti-Ig) from Cappel, Pennsylvania. Lymph node cells or splenocytes were incubated for 15 min at room temperature at lo7 cells/ml of appropriately diluted antibody. They were then washed and incubated an additional 30 min at 37°C in
FOSTER-NURSING
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177
diluted human complement which had been prescreened for low background cytotoxicity. Popliteal Lymph Node Assay A local assay, based on the difference in weight between popliteal lymph nodes (PLN), was carried out in adult F 1 mice ( 10). Seven X 1O6adult spleen cells from F 1 donors were inoculated in the right footpad of sex- and age-matched identical or reciprocal Fl adult hosts. The contralateral footpad received culture medium. Seven days later, F 1 mice were killed and both PLN were excised, trimmed of accompanying tissues, and weighed immediately to an accuracy of 0.01 mg. When indicated, FI hosts received 7 X 1O6splenocytes from foster-nursed F 1 hybrids in the right footpad and the same number of cells from Fl hybrids nursed by their own mothers in the left one. Proliferation Assays A method described by Chaouat and Chaffaux (11) to test the proliferative GvH capacity was used. Mice were inoculated in the right footpad with splenocytes from reciprocal Fl hybrids and in the left footpad with the same number of splenocytes from identical hybrids. On Day 3, both PLN were excised and disrupted and, after two washes, cells were resuspended in a total volume of 1 ml per lymph node with RPM1 1640 supplemented with 10% FCS, antibiotics, and 2 X 10e5M 2-mercaptoethanol. Each suspension was seededin 200-~1aliquots in 96-well Falcon microtiter plates, and 1.O PC1of tritiated thymidine ([3H]TdR; spe activ, 20 Ci/rruV; New England Nuclear Corp., Boston, MA) was added to each well. Cultures were harvested after an 18-hr pulse with a multi-well automated sample harvester and radioactivity was counted in a Packard 3002 liquid scintillation counter. The total counts per minute (cpm) of the five wells were tabulated as the proliferative response of the tested lymph node. Presentation of Data and Statistics Figures and tables contain representative results from one experiment. All experiments were independently repeated three to four times. Student’s t test was used to assessthe statistical significance of the results. RESULTS PLNE Induced by Splenocytesfrom Fl Hybrids in Reciprocal FI Hosts Adult Fl mice of both sexes were inoculated in the right footpad with 7 X lo6 splenocytes from either identical or reciprocal age- and sex-matched Fl hybrids; culture medium was injected in the contralateral footpad. The results obtained are shown in Table 1. As can be observed, the inoculation of splenocytes from Fl hybrids identical to the host did not induce a significant PLNE. On the other hand, the inoculation of (BALB/cXAKR.M/Sn)Fl splenocytes in (AKR.M/SnXBALB/c)Fl hosts, and of (AKR.M/SnXBALB/c)Fl splenocytes in (BALB/cXAKR.M/Sn)Fl hosts induced a significant PLNE. Similar results were obtained when splenocytes from (BALB/cXAKR)Fl , (AKRXBALB/c)Fl , (BALB/cXDBA/2)Fl, and (DBA/
178
NEPOMNASCHY TABLE
ET AL. 1
PLNE Induced by Lyt-l+ T Splenocytes from Fl Hybrids in Reciprocal Fl Hosts
Donor” (AKR.M/Sn X BALB/c)Fl (BALB/c X AKR.M/Sn)Fl (BALB/c X AKR.M/Sn)Fl (AKR.M/Sn X BALB/c)Fl AKR.M/Sn BALB/c (AKR X BALB/c)Fl (BALB/c X AKR)Fl (BALB/c X AKR)Fl (AKR X BALB/c)Fl (BALB/c X AKR)Fl ’ (AKR X BALB/c)Fl ’ (BALB/c x AKR)Fl’ (AKR X BALB/c)Fl ’ (AKR X BALB/c)Fl= (AKR X BALB/c)Flf (AKR X BALB/c)Fl g (DBA/Z X BALB/c)Fl (BALB/c X DBA/2)Fl (BALB/c X DBA/Z)Fl (DBA/2 x BALB/c)Fl (DBA/2 X AKR)Fl (AKR X DBA/Z)Fl (AKR X DBA/Z)Fl (DBA/2 X AKR)Fl (AKR X AKR.M/Sn)Fl (AKR.M/Sn X AKR)Fl (AKR.M/Sn X AKR)Fl (AKR X AKR.M/Sn)Fl
Host (AKR.M/Sn X BALB/c)Fl (AKR.M/Sn X BALB/c)Fl (BALB/c X AKR.M/Sn)Fl (BALB/c X AKR.M/Sn)Fl (BALB/c X AKR.M/Sn)Fl (BALB/c X AKR.M/Sn)Fl (AKR X BALB/c)Fl (AKR X BALB/c)Fl (BALB/c X AKR)Fl (BALB/c x AKR)Fl (BALB/c X AKR)Fl (BALB/c X AKR)Fl (BALB/c X AKR)Fl (BALB/c X AKR)Fl (BALB/c x AKR)Fl (BALB/c X AKR)Fl (BALB/c X AKR)Fl (DBA/2 X BALB/c)FI (DBA/2 X BALB/c)FI (BALB/c X DBA/2)Fl (BALB/c x DBA/2)Fl (DBA/2 X AKR)Fl (DBA/2 X AKR)Fl (AKR X DBA/2)F 1 (AKR X DBA/2)Fl (AKR X AKR.M/Sn)Fl (AKR X AKR.M/Sn)Fl (AKR.M/Sn X AKR)Fl (AKR.M/Sn X AKR)Fl
PLNE b ma + SE (n)’ 0.16+0.13(10) 4.06 + 0.77 (8)* 0.20~0.12(10) 1.49+0.18(12)* 4.90 k 0.27 (6) 2.30 + 0.20 (6) 0.23 + 0.19 (6) 3.63 f 0.28 (8)* 0.32 + 0.18 (6) 3.13*0.42(13)* 0.14+0.10(5) 0.19-cO.l2(5) 0.41 f 0.18 (6) 3.20 f 0.29 (6)* 3.14 f 0.21 (6)* 0.37 f 0.14 (6) 0.3 1 f 0.23 (6) 0.23 zk 0.10 (9) 2.12-cO.73(12)* 0.28 + 0.15 (8) 0.95 + 0.08 (lo)* 0.41 f 0.18 (12) 0.38 + 0.19 (12) 0.32?0.15(12) 0.39 ?z0.20 (12) 0.35 + 0.19 (4) 0.40 + 0.20 (4) 0.29+0.15(5) 0.32 + 0.16 (6)
a 7 X lo6 splenocytes inoculated in the right footpad ofadult sex-matched Fl hosts; culture medium was inoculated in the left footpad. ’ Difference in weight between both popliteal lymph nodes expressed in mg k SE (number of mice). ’ 7 X 1O6donor splenocytes pretreated with anti-Thy 1.2 serum plus complement. d 2 X lo6 donor NWNA splenocytes. ’ 7 X lo6 donor splenocytes pretreated with anti-Lyt-2 serum plus complement. f 7 X lo6 donor splenocytes pretreated with anti-Lyt- 1 serum plus complement. g 7 X lo6 donor splenocytes pretreated with mitomycin C. Note. *, Significant differences, as determined by Student’s t test.
2XBALB/c)F 1 were inoculated in reciprocal F 1 hosts. Figure 1 shows representative results obtained when dose-response curves were performed. As can be observed, while the inoculation of splenocytes from Fl hybrids identical to the hosts was able to induce a slight PLNE only when high doses of cells were inoculated, a significant and always higher PLNE was obtained even when low doses of splenocytes from Fl reciprocal hybrids were grafted. No differences between sexeswere observed. On the other hand, when reciprocal Fl pairs between AKR and DBA/2, and AKR and AKR.M/Sn parental strains were used, no significant PLNE was observed when splenocytes were inoculated in reciprocal Fl hosts (Table 1).
FOSTER-NURSING AND PLNE IN SYNGENEIC HOSTS
179
PLNElmg)
5r
SPLENOCYTES
x lKb
FIG. 1. PLNE in (BALB/cXAKR)Fl hosts inoculated in the right footpad with different dosesof splenocytes from (BALB/cxAKR)Fl or (AKRxBALB/c)Fl; culture medium was inoculated in the left footpad. Each point represents the arithmetic mean of the differences in weight between right and left PLN of six to eight mice, expressed in milligrams. SE were lessthan 10%.
Lyt 1’ T Fl SplenocytesAre Responsiblefor PLNE Induction in Reciprocal Fl Hosts The inoculation of Fl NWNA splenocytesled to results similar to those obtained with unfractionated splenocytes,while the pretreatment of donor cellswith anti-Thy 1.2or antiLyt 1 serum plus complement wasableto abrogatePLNE (Table 1).Pretreatmentof donor splenocyteswith anti-Lyt 2 serum plus complement did not signihcantly alter PLNE. Mitomycin C pretreatment of donor cells also abrogatedPLNE. Cell Proliferation in PLN Table 2 depicts a typical experiment where DNA synthesis in PLN was measured 3 days after Fl hybrids had received 7 X 1O6splenocytes from the reciprocal F 1 combination in the right footpad and the same number of splenocytes from identical Fl hybrids in the contralateral footpad. As can be observed, PLN nodes from (BALB/ cXAKR)Fl and (AKRXBALB/c)Fl grafted with splenocytes from reciprocal Fl hybrids, showed significant increases in proliferative levels as compared with proliferation observed in PLN draining the inocula of splenocytes from identical Fl hybrids (proliferative ratios ranged between 8 and 14). On the other hand, PLN from (AKRXAKR.M/Sn)Fl, (AKR.M/SnXAKR)Fl, (DBA/2XAKR)Fl, and (AKRXDBA/ 2)Fl grafted with splenocytes from reciprocal Fl hybrids, did not show increases in [3H]TdR incorporation. As can be seen in Table 3, the inoculation of (BALB/cXAKR)Fl hybrids with NWNA splenocytes from (AKRXBALB/c)Fl, led to results similar to those obtained with unfractionated splenocytes, while the pretreatment of donor cells with anti-Thy 1.2 or anti-Lyt 1 serum plus complement was able to abrogate the increases in PLN proliferation. Pretreatment of donor splenocytes with anti-Lyt 2 serum plus complement did not alter significantly PLN proliferation. When 2 X lo6 NWNA splenocytes from (AKRXBALB/c)Fl pretreated with anti-Lyt 2 serum plus complement were inoculated in (BALB/cXAKR)Fl and, at Day 3, lymph nodes were treated with antiIg serum plus complement before the addition of [3H]TdR, the proliferative response of the PLN was significantly decreased.When donor NWNA splenocytes were pre-
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NEPOMNASCHY
ET AL.
TABLE 2 Proliferation
Host
of PLN Draining the Inocula of Splenocytes from Reciprocal Fl Hybrids Grafted cells” right footpad
(AKR X BALB/c)Fl
(BALB/c
(BALB/c
X AKR)Fl
(AKR X BALB/c)Fl
(BALB/c
X AKR)Fl
BALBfc
X AKR)Fl
(AKR X DBA/Z)Fl
(DBA/2
(DBA/Z X AKR)Fl
(AKR X DBA/Z)Fl
X AKR)Fl
wm 17,325 19,249 35,406 25,036 33,207 64,644 67,574 54,939 100,926 117,717 90,560 4,571 4,962 7,923 6,290 3,974 7,420 5,69 1 9,238
Grafted cells” left footpad (AKR X BALB/c)F 1
(BALB/c
X AKR)Fl
(BALB/c
X AKR)Fl
(AKR X DBA/2)Fl
(DBA/Z X AKR)Fl
cm 1700 1920 2384 2518 3888 6540 5674 4309 4519 495 1 2120 3672 5489 6832 4720 3160 7108 5986 8734
Ratio b 9.2 10.0 14.4 10.0 8.5 9.9 11.9 12.7 22.3 23.8 42.7 1.2 0.9 1.2 1.5 1.3 1.0 0.9 1.0
0 7 X lo6 splenocytes inoculated in the footpad of adult sex-matched Fl hosts. On Day 3, the PLN nodes were excised and disrupted. The obtained cell suspensions were harvested after an 18-hr pulse of [3H]TdR. b cpm right PLN/cpm left PLN.
treated with anti-Lyt 2 serum plus complement and the excised PLN were treated either with anti-Lyt 2 or anti-Lyt 1 serum plus complement, C3H]TdR incorporation was also significantly decreased(Table 3).
Efect of Foster-Nursing on the Ability of Splenocytesto Induce PLNE Fl mice were foster-nursed on mothers from the paternal strain and, when adults, their splenocytes were inoculated in the right footpad of either identical or reciprocal F 1 hosts; splenocytes from normally nursed F 1 mice identical to the host were inoculated in the contralateral footpad. As can be seenin Fig. 2, the inoculation of splenoFl in (AKRXBALB/c)Fl hosts induced a significytes from (AKRXBALB/c),,,, cant PLNE. On the contrary, when inoculated in (BALB/cXAKR)Fl hosts, no PLNE could be registered. Similarly, when splenocytes from (BALB/cXAKR)~*~FI hybrids were used as donors, a significant lymph node enlargement could be registered in (BALB/cXAKR)Fl hosts but not in (AKRXBALB/c)Fl hosts. Similar results were obtained with (BALB/cXDBA/~)~~*,~ Fl hybrids (Fig. 2). As in the normally nursed Fl hybrids, Lyt I+ T splenocytes were responsible for the induction of PLNE (Fig. 2). Proliferative events occurring in lymph nodes draining the inoculum of splenocytes from foster-nursed F 1 hybrids were found to be similar to those observed with splenocytes from normally nursed hybrids (Fig. 3). That is to say, foster-nursing on mothers from the paternal strain was able to alter the ability of spleen cells to induce PLNE, increasing their ability to induce lymph
FOSTER-NURSING AND PLNE IN SYNGENEIC HOSTS
181
TABLE 3 Phenotype Determination of Inducer and Proliferating Cells Graft pretreatment’
PLN pretreatment b
-
Anti-Thy 1.2 Anti-Lyt 1
-
Anti-Lyt 2
-
NWNA + Anti-Lyt 1
Anti-Ig
NWNA + Anti-Lyt 1
Anti-Lfl 1
NWNA + Anti-Lyt 1
Anti-Lyt 2
Right PLN cpm
Left PLN wm
Ratio’
15,286 21,143 16,639 24,277 2,343 3,228 1,835 2,549 1,678 4,566 2,455 18,343 20,255 18,311 25,941 6,047 3,517 9,520 2,620 7,391 4,606 5,229 10,375 3,270 8,606 8,012 7,694
2175 2405 1547 1323 2455 3034 1753 2458 1356 4233 2156 2549 1934 1495 2130 1705 1654 2150 1943 1391 1609 1104 2606 826 1831 2613 1476
7.0 8.8 10.7 18.3 0.9 1.0 1.0 1.0 1.2 1.0 1.1 7.2 10.5 12.2 12.2 3.5 2.1 4.4 2.9 5.3 2.9 4.7 3.7 4.0 4.7 3.1 5.2
a (BALB/c X AKR)Fl hosts were inoculated in the right footpad with 7 X lo6 splenocytes or 2 X lo6 NWNA splenocytes from (AKR X BALB/c)Fl hybrids, pretreated with the appropriate antibody plus complement. The left footpad was inoculated with 7 X lo6 (BALB/c X AKR)Fl splenocytes. ’ On Day 3, PLN were excised, disrupted, and treated with the appropriate antibody plus complement. The obtained cell suspensionswere harvested after an 18-hr pulse of [‘H]TdR. ’ cpm right PLN/cpm left PLN.
node enlargement in identical Fl hosts and abrogating their capacity to induce this reaction when inoculated in reciprocal Fl hosts. The effect of foster nursing could be registered even when B- to 12-month-old F 1 hybrids were used. On the other hand, when (AKRXDBA/2)Fl hybrids were foster-nursed on mothers from the paternal strain, no changesin the ability to induce PLNE were observed (Fig. 2). Similar results were obtained with foster-nursed (AISRXAKB.M/Sn)Fl hybrids (data not shown). Neither was the ability to induce proliferation altered by foster-nursing (Fig. 3). DISCUSSION We had previously reported that splenocytes from reciprocal Fl hybrids differed in their ability to induce T cell proliferation in syngeneic mixed lymphocyte reac-
182
NEPOMNASCHY HOST
@KP. x EUWc)Fl @LB/c
Pm
GRAFT
GRAFT PRETR.
x mlB/c)~,c
---
II
x MR)Fl
(Mb? x WB/c)Fl
@.UI/C
ET AL.
___ NWNA
x WWF1
WKR x BALB/c)Fl !WB/C
X WZ)Fl
KXS/Z
x ENB/c)Fl
(m/C
X cBA/2)w,Fl
---
(PkR x BWFl (M/2x
MR)FI
PLNE(mp)
FIG. 2. PLNE of Fl hosts inoculated in the right footpad with 7 X IO6splenocytes or 2 X lo6 NWNA cells from foster-nursed Fl hybrids. When indicated, splenocytes or NWNA cells were pretreated with the appropriate antibody plus complement. In the contralateral footpad, hosts received the same number of splenocytes from identical FI hybrids. Each bar represents the arithmetic mean of the difference in weight between both PLN of six to eight mice, expressed in milligrams. SE was lessthan 10%.
tions, being lactation centrally involved. We had postulated (4) that the antigenic impact of self-histocompatibility antigens (MIS included) present on maternal milk cells, could be responsible for the differences registered. The results reported herein show that splenocytes from Fl hybrids from BALB/c and AKR, BALB/c and AKR.M/Sn, and BALB/c and DBA/2 parental strains, are able to induce a significant PLNE when inoculated in the footpad of sex- and agematched reciprocal Fl hosts. Foster-nursing of F 1 hybrids on mothers from the paternal strain was able to alter the ability of splenocytes to induce this reaction. The magnitude of the syngeneic PLNE observed was comparable to that of graft vs host reactions induced with the parental strains. Pretreatment of grafted cells with mitomycin C abrogated the PLNE. The treatment of donor cells with anti-Thy or anti-Lyt 1 plus complement but not with anti-Lyt 2 abrogated the reaction, while nylon wool nonadherent splenocytes led to results similar to those obtained with the whole spleen population. These results suggestthat donor Lyt I+ T cells are the cells inducing this reaction. To evaluate the proliferation occurring in the PLN, an in vim-in vitro method (11) was used. Cells from the PLN were harvested and pulsed in vitro with [3H]TdR for measurement of proliferation. The PLN draining the inocula of Lyt l+ splenocytes from reciprocal hybrids showed proliferative levels significantly higher than those induced by splenocytes from identical hybrids, indicating that in situ proliferation accompanied the PLNE observed. Proliferative levels registered were consistently lower than those induced by inoculation of parental splenocytes. When assayswere carried out inoculating T or Lyt l+ splenocytes and treating cells from the draining popliteal lymph nodes with anti-Lyt 1, anti-Lyt 2, or anti-Ig serum plus complement,
FOSTER-NURSING AND PLNE IN SYNGENEIC HOSTS GRAFT
HOST
(PKR x fVWc)Fl
wB/c
183
GRAFT PRETR.
(AKR x WIB/C)~,~F~
x pKR)Fl ml4
(AKR x WLEI/c)Fl
Thy 1.2 /I
(.WR.M/SnxMR)Fl (MR x MR.WSn)Fl
Lyt 1
v'xR.wsn
x PxR)fM II
RATIO
(qmr@tkpnleft)
FIG. 3. [‘H]TdR incorporation by PLN cells from Fl hosts inoculated in the right footpad with 7 X IO6 splenocytes or NWNA cells from foster-nursed Fl hybrids. When indicated, donor cells were treated with the appropriate antibody plus complement. In the contralateral footpad, hosts received the same number of splenocytes from identical Fl hybrids. On Day 3, the PLN nodes were excised, disrupted, and treated with the appropriate antibody plus complement. The obtained cell suspensionswere harvested after an IShr pulse of i3HJTdR. Each bar represents the arithmetic mean of cpm right PLN/cpm left PLN (number of mice, four). SE were lessthan 15%.
in vitro proliferation was significantly decreased,indicating that proliferation of host Lyt 2+ and B cells is involved. It remains unknown whether Lyt l+ cells proliferating on Day 3 are of host or graft origin. Foster-nursing of Fl hybrids on mothers from the paternal strain was able to induce permanent alterations in the ability of splenocytes to induce PLNE: a significant PLNE could be detected when T or Lyt I+ splenocytes from foster-nursed Fl hybrids were grafted in identical normally nursed Fl hosts; on the contrary, no PLNE was detected when splenocytes from foster-nursed F 1 hybrids were inoculated in reciprocal normally nursed Fl hosts. Thus, the pattern of spleen ability to induce PLNE of foster-nursed Fl mice resembled that observed in the reciprocal Fl combination nursed by its own mother. Proliferative events occurring in lymph nodes draining the inoculum of splenocytes from foster-nursed Fl hybrids were found to be similar to those observed with splenocytes from normally nursed reciprocal hybrids. These results suggestthat lactation would be a critical event determining the ability of T splenocytes to induce PLNE when inoculated in identical Fl hosts. When strain combinations between parental strains sharing Mls” antigens were used, neither significant PLNE nor increases in lymph node proliferation could be registered. Foster-nursing of these Fl hybrids did not alter the ability of their splenocytes to induce PLNE. The nature of the alterations reported herein still remains unknown. The induction of qualitative antigenic differences through milk, i.e., the appearance of altered selfcomponents by an endogenous virus cannot be discarded. However, other possibili-
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NEPOMNASCHY
ET AL,
ties may be considered. The fact that the effect of lactation could be detected in strain combinations involving one parental strain expressing the stimulatory Mls” allele and the other parental strain bearing the nonstimulatory Mlsb allele but could not be detected in those sharing Mls” determinants, raises the possibility that these antigens could be involved. It has been demonstrated that antigenic impacts in newborn mice are able to induce permanent alterations in their immune responsiveness (12-14). Both Mls and Ia antigens seem to be poorly expressed in newborn mice ( 15- 17). On the other hand, it has been recently reported ( 16) that self-reactive (V,‘,) T cells with a mature CD4+ surface phenotype are present during the early postnatal period in the thymus of Mls” mice and that a small portion of these cells can even be detected in the mesenteric lymph nodes of 2- to 3-day-old mice. If, as it has been reported (2), milk cells are able to penetrate in the newborn circulation, the possibility exists that the confrontation between these self-reactive cells and maternal Mls antigens could influence the T cell repertoire of the litter. ACKNOWLEDGMENTS We thank Mr. Juan Portaluppi and Mr. Antonio Morales for efficient technical assistance. This work was made possible by grants from CONICET.
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IMMUNOLOGY
128, 175%184(1990)
Popliteal Lymph Node Enlargement Induced in Syngeneic Hosts by T Cells from Foster-Nursed Mice IRENENEPOMNASCHY,‘ADRIANAD~ROCHE,’SANDRATORELLO,’ ALEJANDRAGOLDMAN,VALERIA BUGGIANO,* CHRISTIANEDOSNEPASQUALINI,’ ANDISABELPIAZZON’ Divisicin Medicina Experimental, Instituto de Investigaciones Hematol6gicas. Academia National de Medicina, Las Heras 3092, 1425 Buenos Aires, Argentina Received October 5. 1989; acceptedJanuary 13, 1990 Splenocytes from adult Fl mice were assayed for their capacity to induce popliteal lymph node enlargement (PLNE) when inoculated in the footpad of identical or reciprocal FI hosts. The results obtained showed that: (i) T Lyt l+ splenocytes from adult Fl hybrids were able to induce a significant PLNE when inoculated in reciprocal but not in identical Fl hosts.(ii) Fosternursing of Fl hybrids on mothers from the paternal strain was able to induce permanent alterations in the ability of their T splenocytes to induce PLNE: Lyt l+ splenocytes were able to induce significant PLNE in identical but not in reciprocal Fl hosts.Thus, the ability of T splenocytes from foster-nursed Fl hybrids to induce PLNE resembled that observed in reciprocal Fl hybrids nursed by their own mothers. (iii) PLNE was accompanied by cell proliferation involving host B and T lymphocytes. (iv) This PLNE could be detected using Fl hybrids from parental strains differing or not at H-2 antigens but involving a parental strain expressing the stimulatory MIS” allele and a parental strain bearing the nonstimulatory Mlsb allele while it was not observed in F 1 hybrids from parental strains sharing MIS” antigens. o 1990 Academic PXSS, hc.
INTRODUCTION Matemofetal relationship involves not only pregnancy but also breast feeding. Colostrum and milk are rich in immunoglobulins, macrophages, and T and B lymphocytes (1). Weiler et al. (2) have reported that milk cells may penetrate the stomach wall and colonize the neonatal rodents. It has been proposed that the exposure of the neonate to maternal antigens during breast-feeding may alter the immunologic reactivity of the litter toward maternal allografts (3). In a previous paper (4) we have reported that splenocytes from reciprocal Fl hybrids differ in their ability to stimulate T cell proliferation in syngeneic mixed lymphocyte reactions. Foster-nursing of Fl hybrids on mothers from the paternal strain was able to induce permanent alterations in the ability of their splenocytes to stimulate T cell proliferation in these reactions. The stimulatory ability of splenocytes from r Member of the Research Career of the Consejo National de Investigaciones Cientificas y Tknicas, CONICET. * Fellowship of CONICET. 175 0008-8749/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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foster-nursed hybrids became indistinguishable from that observed in the reciprocal Fl combination nursed by its own mother. Localized proliferative in vivo responsesto subcutaneously injected syngeneic cells have previously been described. Popliteal lymph node enlargement (PLNE) has been induced in normal mice by inoculating syngeneic T cells from aged mice (5), and neonatally (6) or adult (7) thymectomized mice. L’Age-Stehr and Diamantstein have reported (8) that splenocytes from cyclophosphamide-pretreated mice were also able to induce PLNE when inoculated in normal syngeneic mice. In this paper, we show that lactation is responsible for the ability of Lyt 1+ T splenocytes from Fl hybrids to induce a significant PLNE when inoculated in reciprocal Fl hosts. MATERIALS AND METHODS Mice The following pairs of reciprocal Fl hybrids, raised in our colony were used: (BALB/cXAKR)Fl (H-2dXH-2k) (MLSbXMlsa) and (AKRXBALB/c)Fl; (BALB/ cXAKR.M/Sn)Fl (H-2dXH-2m) (MlsbXMlsa) and (AKR.M/SnXBALB/c)Fl; (BALB/cXDBA/2)Fl (H-2dXH-2d) (MlsbXMls”) and (DBA/2XBALB/c)Fl; (AKRXDBA/2)Fl (H-2kXH-2d) (Mls”XMls”) and (DBA/2XAKR)Fl; (AISRXAKR.M/ Sn)Fl (H-2kXH-2m) (Mls”XMls”) and (AKR.M/SnXAKR)Fl. Animals from both sexesand 2- to 4-months old were used. The maternal strain of the F 1 hybrid is always denoted first. Foster-Nursing Full-term pregnant females were placed in special cagesto avoid any suckling by the litter; immediately after birth, half of the litter was returned to its own mother and the other half was foster-nursed on mothers from the paternal strain which had been pregnant from syngeneic males. Foster-nursed mice were denoted as (AXB)fBFl. They were used when they were adults (2-4 months) and, in some experiments, when 8- to 12-month old. Cell Suspensions Splenocytes were obtained aseptically by gently pressing the spleen through a stainless-steel mesh. Viability was assessedby trypan blue exclusion (over 90% was required for use) after which the cells were resuspended at the desired concentration in RPM1 1640 (GIBCO, Grand Island, NY)). Spleen populations enriched in T cells (NWNA) were obtained by passagethrough nylon wool columns as described by Julius et al. (9). Antisera Treatment The anti-Thy 1.2., anti-Lyt 1, and anti-Lyt 2 sera were obtained from BectonDickinson Labware, Oxnard; goat anti-mouse immunoglobulins (IgE+IgG+IgM) (H&L) (anti-Ig) from Cappel, Pennsylvania. Lymph node cells or splenocytes were incubated for 15 min at room temperature at lo7 cells/ml of appropriately diluted antibody. They were then washed and incubated an additional 30 min at 37°C in
FOSTER-NURSING
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diluted human complement which had been prescreened for low background cytotoxicity. Popliteal Lymph Node Assay A local assay, based on the difference in weight between popliteal lymph nodes (PLN), was carried out in adult F 1 mice ( 10). Seven X 1O6adult spleen cells from F 1 donors were inoculated in the right footpad of sex- and age-matched identical or reciprocal Fl adult hosts. The contralateral footpad received culture medium. Seven days later, F 1 mice were killed and both PLN were excised, trimmed of accompanying tissues, and weighed immediately to an accuracy of 0.01 mg. When indicated, FI hosts received 7 X 1O6splenocytes from foster-nursed F 1 hybrids in the right footpad and the same number of cells from Fl hybrids nursed by their own mothers in the left one. Proliferation Assays A method described by Chaouat and Chaffaux (11) to test the proliferative GvH capacity was used. Mice were inoculated in the right footpad with splenocytes from reciprocal Fl hybrids and in the left footpad with the same number of splenocytes from identical hybrids. On Day 3, both PLN were excised and disrupted and, after two washes, cells were resuspended in a total volume of 1 ml per lymph node with RPM1 1640 supplemented with 10% FCS, antibiotics, and 2 X 10e5M 2-mercaptoethanol. Each suspension was seededin 200-~1aliquots in 96-well Falcon microtiter plates, and 1.O PC1of tritiated thymidine ([3H]TdR; spe activ, 20 Ci/rruV; New England Nuclear Corp., Boston, MA) was added to each well. Cultures were harvested after an 18-hr pulse with a multi-well automated sample harvester and radioactivity was counted in a Packard 3002 liquid scintillation counter. The total counts per minute (cpm) of the five wells were tabulated as the proliferative response of the tested lymph node. Presentation of Data and Statistics Figures and tables contain representative results from one experiment. All experiments were independently repeated three to four times. Student’s t test was used to assessthe statistical significance of the results. RESULTS PLNE Induced by Splenocytesfrom Fl Hybrids in Reciprocal FI Hosts Adult Fl mice of both sexes were inoculated in the right footpad with 7 X lo6 splenocytes from either identical or reciprocal age- and sex-matched Fl hybrids; culture medium was injected in the contralateral footpad. The results obtained are shown in Table 1. As can be observed, the inoculation of splenocytes from Fl hybrids identical to the host did not induce a significant PLNE. On the other hand, the inoculation of (BALB/cXAKR.M/Sn)Fl splenocytes in (AKR.M/SnXBALB/c)Fl hosts, and of (AKR.M/SnXBALB/c)Fl splenocytes in (BALB/cXAKR.M/Sn)Fl hosts induced a significant PLNE. Similar results were obtained when splenocytes from (BALB/cXAKR)Fl , (AKRXBALB/c)Fl , (BALB/cXDBA/2)Fl, and (DBA/
178
NEPOMNASCHY TABLE
ET AL. 1
PLNE Induced by Lyt-l+ T Splenocytes from Fl Hybrids in Reciprocal Fl Hosts
Donor” (AKR.M/Sn X BALB/c)Fl (BALB/c X AKR.M/Sn)Fl (BALB/c X AKR.M/Sn)Fl (AKR.M/Sn X BALB/c)Fl AKR.M/Sn BALB/c (AKR X BALB/c)Fl (BALB/c X AKR)Fl (BALB/c X AKR)Fl (AKR X BALB/c)Fl (BALB/c X AKR)Fl ’ (AKR X BALB/c)Fl ’ (BALB/c x AKR)Fl’ (AKR X BALB/c)Fl ’ (AKR X BALB/c)Fl= (AKR X BALB/c)Flf (AKR X BALB/c)Fl g (DBA/Z X BALB/c)Fl (BALB/c X DBA/2)Fl (BALB/c X DBA/Z)Fl (DBA/2 x BALB/c)Fl (DBA/2 X AKR)Fl (AKR X DBA/Z)Fl (AKR X DBA/Z)Fl (DBA/2 X AKR)Fl (AKR X AKR.M/Sn)Fl (AKR.M/Sn X AKR)Fl (AKR.M/Sn X AKR)Fl (AKR X AKR.M/Sn)Fl
Host (AKR.M/Sn X BALB/c)Fl (AKR.M/Sn X BALB/c)Fl (BALB/c X AKR.M/Sn)Fl (BALB/c X AKR.M/Sn)Fl (BALB/c X AKR.M/Sn)Fl (BALB/c X AKR.M/Sn)Fl (AKR X BALB/c)Fl (AKR X BALB/c)Fl (BALB/c X AKR)Fl (BALB/c x AKR)Fl (BALB/c X AKR)Fl (BALB/c X AKR)Fl (BALB/c X AKR)Fl (BALB/c X AKR)Fl (BALB/c x AKR)Fl (BALB/c X AKR)Fl (BALB/c X AKR)Fl (DBA/2 X BALB/c)FI (DBA/2 X BALB/c)FI (BALB/c X DBA/2)Fl (BALB/c x DBA/2)Fl (DBA/2 X AKR)Fl (DBA/2 X AKR)Fl (AKR X DBA/2)F 1 (AKR X DBA/2)Fl (AKR X AKR.M/Sn)Fl (AKR X AKR.M/Sn)Fl (AKR.M/Sn X AKR)Fl (AKR.M/Sn X AKR)Fl
PLNE b ma + SE (n)’ 0.16+0.13(10) 4.06 + 0.77 (8)* 0.20~0.12(10) 1.49+0.18(12)* 4.90 k 0.27 (6) 2.30 + 0.20 (6) 0.23 + 0.19 (6) 3.63 f 0.28 (8)* 0.32 + 0.18 (6) 3.13*0.42(13)* 0.14+0.10(5) 0.19-cO.l2(5) 0.41 f 0.18 (6) 3.20 f 0.29 (6)* 3.14 f 0.21 (6)* 0.37 f 0.14 (6) 0.3 1 f 0.23 (6) 0.23 zk 0.10 (9) 2.12-cO.73(12)* 0.28 + 0.15 (8) 0.95 + 0.08 (lo)* 0.41 f 0.18 (12) 0.38 + 0.19 (12) 0.32?0.15(12) 0.39 ?z0.20 (12) 0.35 + 0.19 (4) 0.40 + 0.20 (4) 0.29+0.15(5) 0.32 + 0.16 (6)
a 7 X lo6 splenocytes inoculated in the right footpad ofadult sex-matched Fl hosts; culture medium was inoculated in the left footpad. ’ Difference in weight between both popliteal lymph nodes expressed in mg k SE (number of mice). ’ 7 X 1O6donor splenocytes pretreated with anti-Thy 1.2 serum plus complement. d 2 X lo6 donor NWNA splenocytes. ’ 7 X lo6 donor splenocytes pretreated with anti-Lyt-2 serum plus complement. f 7 X lo6 donor splenocytes pretreated with anti-Lyt- 1 serum plus complement. g 7 X lo6 donor splenocytes pretreated with mitomycin C. Note. *, Significant differences, as determined by Student’s t test.
2XBALB/c)F 1 were inoculated in reciprocal F 1 hosts. Figure 1 shows representative results obtained when dose-response curves were performed. As can be observed, while the inoculation of splenocytes from Fl hybrids identical to the hosts was able to induce a slight PLNE only when high doses of cells were inoculated, a significant and always higher PLNE was obtained even when low doses of splenocytes from Fl reciprocal hybrids were grafted. No differences between sexeswere observed. On the other hand, when reciprocal Fl pairs between AKR and DBA/2, and AKR and AKR.M/Sn parental strains were used, no significant PLNE was observed when splenocytes were inoculated in reciprocal Fl hosts (Table 1).
FOSTER-NURSING AND PLNE IN SYNGENEIC HOSTS
179
PLNElmg)
5r
SPLENOCYTES
x lKb
FIG. 1. PLNE in (BALB/cXAKR)Fl hosts inoculated in the right footpad with different dosesof splenocytes from (BALB/cxAKR)Fl or (AKRxBALB/c)Fl; culture medium was inoculated in the left footpad. Each point represents the arithmetic mean of the differences in weight between right and left PLN of six to eight mice, expressed in milligrams. SE were lessthan 10%.
Lyt 1’ T Fl SplenocytesAre Responsiblefor PLNE Induction in Reciprocal Fl Hosts The inoculation of Fl NWNA splenocytesled to results similar to those obtained with unfractionated splenocytes,while the pretreatment of donor cellswith anti-Thy 1.2or antiLyt 1 serum plus complement wasableto abrogatePLNE (Table 1).Pretreatmentof donor splenocyteswith anti-Lyt 2 serum plus complement did not signihcantly alter PLNE. Mitomycin C pretreatment of donor cells also abrogatedPLNE. Cell Proliferation in PLN Table 2 depicts a typical experiment where DNA synthesis in PLN was measured 3 days after Fl hybrids had received 7 X 1O6splenocytes from the reciprocal F 1 combination in the right footpad and the same number of splenocytes from identical Fl hybrids in the contralateral footpad. As can be observed, PLN nodes from (BALB/ cXAKR)Fl and (AKRXBALB/c)Fl grafted with splenocytes from reciprocal Fl hybrids, showed significant increases in proliferative levels as compared with proliferation observed in PLN draining the inocula of splenocytes from identical Fl hybrids (proliferative ratios ranged between 8 and 14). On the other hand, PLN from (AKRXAKR.M/Sn)Fl, (AKR.M/SnXAKR)Fl, (DBA/2XAKR)Fl, and (AKRXDBA/ 2)Fl grafted with splenocytes from reciprocal Fl hybrids, did not show increases in [3H]TdR incorporation. As can be seen in Table 3, the inoculation of (BALB/cXAKR)Fl hybrids with NWNA splenocytes from (AKRXBALB/c)Fl, led to results similar to those obtained with unfractionated splenocytes, while the pretreatment of donor cells with anti-Thy 1.2 or anti-Lyt 1 serum plus complement was able to abrogate the increases in PLN proliferation. Pretreatment of donor splenocytes with anti-Lyt 2 serum plus complement did not alter significantly PLN proliferation. When 2 X lo6 NWNA splenocytes from (AKRXBALB/c)Fl pretreated with anti-Lyt 2 serum plus complement were inoculated in (BALB/cXAKR)Fl and, at Day 3, lymph nodes were treated with antiIg serum plus complement before the addition of [3H]TdR, the proliferative response of the PLN was significantly decreased.When donor NWNA splenocytes were pre-
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TABLE 2 Proliferation
Host
of PLN Draining the Inocula of Splenocytes from Reciprocal Fl Hybrids Grafted cells” right footpad
(AKR X BALB/c)Fl
(BALB/c
(BALB/c
X AKR)Fl
(AKR X BALB/c)Fl
(BALB/c
X AKR)Fl
BALBfc
X AKR)Fl
(AKR X DBA/Z)Fl
(DBA/2
(DBA/Z X AKR)Fl
(AKR X DBA/Z)Fl
X AKR)Fl
wm 17,325 19,249 35,406 25,036 33,207 64,644 67,574 54,939 100,926 117,717 90,560 4,571 4,962 7,923 6,290 3,974 7,420 5,69 1 9,238
Grafted cells” left footpad (AKR X BALB/c)F 1
(BALB/c
X AKR)Fl
(BALB/c
X AKR)Fl
(AKR X DBA/2)Fl
(DBA/Z X AKR)Fl
cm 1700 1920 2384 2518 3888 6540 5674 4309 4519 495 1 2120 3672 5489 6832 4720 3160 7108 5986 8734
Ratio b 9.2 10.0 14.4 10.0 8.5 9.9 11.9 12.7 22.3 23.8 42.7 1.2 0.9 1.2 1.5 1.3 1.0 0.9 1.0
0 7 X lo6 splenocytes inoculated in the footpad of adult sex-matched Fl hosts. On Day 3, the PLN nodes were excised and disrupted. The obtained cell suspensions were harvested after an 18-hr pulse of [3H]TdR. b cpm right PLN/cpm left PLN.
treated with anti-Lyt 2 serum plus complement and the excised PLN were treated either with anti-Lyt 2 or anti-Lyt 1 serum plus complement, C3H]TdR incorporation was also significantly decreased(Table 3).
Efect of Foster-Nursing on the Ability of Splenocytesto Induce PLNE Fl mice were foster-nursed on mothers from the paternal strain and, when adults, their splenocytes were inoculated in the right footpad of either identical or reciprocal F 1 hosts; splenocytes from normally nursed F 1 mice identical to the host were inoculated in the contralateral footpad. As can be seenin Fig. 2, the inoculation of splenoFl in (AKRXBALB/c)Fl hosts induced a significytes from (AKRXBALB/c),,,, cant PLNE. On the contrary, when inoculated in (BALB/cXAKR)Fl hosts, no PLNE could be registered. Similarly, when splenocytes from (BALB/cXAKR)~*~FI hybrids were used as donors, a significant lymph node enlargement could be registered in (BALB/cXAKR)Fl hosts but not in (AKRXBALB/c)Fl hosts. Similar results were obtained with (BALB/cXDBA/~)~~*,~ Fl hybrids (Fig. 2). As in the normally nursed Fl hybrids, Lyt I+ T splenocytes were responsible for the induction of PLNE (Fig. 2). Proliferative events occurring in lymph nodes draining the inoculum of splenocytes from foster-nursed F 1 hybrids were found to be similar to those observed with splenocytes from normally nursed hybrids (Fig. 3). That is to say, foster-nursing on mothers from the paternal strain was able to alter the ability of spleen cells to induce PLNE, increasing their ability to induce lymph
FOSTER-NURSING AND PLNE IN SYNGENEIC HOSTS
181
TABLE 3 Phenotype Determination of Inducer and Proliferating Cells Graft pretreatment’
PLN pretreatment b
-
Anti-Thy 1.2 Anti-Lyt 1
-
Anti-Lyt 2
-
NWNA + Anti-Lyt 1
Anti-Ig
NWNA + Anti-Lyt 1
Anti-Lfl 1
NWNA + Anti-Lyt 1
Anti-Lyt 2
Right PLN cpm
Left PLN wm
Ratio’
15,286 21,143 16,639 24,277 2,343 3,228 1,835 2,549 1,678 4,566 2,455 18,343 20,255 18,311 25,941 6,047 3,517 9,520 2,620 7,391 4,606 5,229 10,375 3,270 8,606 8,012 7,694
2175 2405 1547 1323 2455 3034 1753 2458 1356 4233 2156 2549 1934 1495 2130 1705 1654 2150 1943 1391 1609 1104 2606 826 1831 2613 1476
7.0 8.8 10.7 18.3 0.9 1.0 1.0 1.0 1.2 1.0 1.1 7.2 10.5 12.2 12.2 3.5 2.1 4.4 2.9 5.3 2.9 4.7 3.7 4.0 4.7 3.1 5.2
a (BALB/c X AKR)Fl hosts were inoculated in the right footpad with 7 X lo6 splenocytes or 2 X lo6 NWNA splenocytes from (AKR X BALB/c)Fl hybrids, pretreated with the appropriate antibody plus complement. The left footpad was inoculated with 7 X lo6 (BALB/c X AKR)Fl splenocytes. ’ On Day 3, PLN were excised, disrupted, and treated with the appropriate antibody plus complement. The obtained cell suspensionswere harvested after an 18-hr pulse of [‘H]TdR. ’ cpm right PLN/cpm left PLN.
node enlargement in identical Fl hosts and abrogating their capacity to induce this reaction when inoculated in reciprocal Fl hosts. The effect of foster nursing could be registered even when B- to 12-month-old F 1 hybrids were used. On the other hand, when (AKRXDBA/2)Fl hybrids were foster-nursed on mothers from the paternal strain, no changesin the ability to induce PLNE were observed (Fig. 2). Similar results were obtained with foster-nursed (AISRXAKB.M/Sn)Fl hybrids (data not shown). Neither was the ability to induce proliferation altered by foster-nursing (Fig. 3). DISCUSSION We had previously reported that splenocytes from reciprocal Fl hybrids differed in their ability to induce T cell proliferation in syngeneic mixed lymphocyte reac-
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NEPOMNASCHY HOST
@KP. x EUWc)Fl @LB/c
Pm
GRAFT
GRAFT PRETR.
x mlB/c)~,c
---
II
x MR)Fl
(Mb? x WB/c)Fl
@.UI/C
ET AL.
___ NWNA
x WWF1
WKR x BALB/c)Fl !WB/C
X WZ)Fl
KXS/Z
x ENB/c)Fl
(m/C
X cBA/2)w,Fl
---
(PkR x BWFl (M/2x
MR)FI
PLNE(mp)
FIG. 2. PLNE of Fl hosts inoculated in the right footpad with 7 X IO6splenocytes or 2 X lo6 NWNA cells from foster-nursed Fl hybrids. When indicated, splenocytes or NWNA cells were pretreated with the appropriate antibody plus complement. In the contralateral footpad, hosts received the same number of splenocytes from identical FI hybrids. Each bar represents the arithmetic mean of the difference in weight between both PLN of six to eight mice, expressed in milligrams. SE was lessthan 10%.
tions, being lactation centrally involved. We had postulated (4) that the antigenic impact of self-histocompatibility antigens (MIS included) present on maternal milk cells, could be responsible for the differences registered. The results reported herein show that splenocytes from Fl hybrids from BALB/c and AKR, BALB/c and AKR.M/Sn, and BALB/c and DBA/2 parental strains, are able to induce a significant PLNE when inoculated in the footpad of sex- and agematched reciprocal Fl hosts. Foster-nursing of F 1 hybrids on mothers from the paternal strain was able to alter the ability of splenocytes to induce this reaction. The magnitude of the syngeneic PLNE observed was comparable to that of graft vs host reactions induced with the parental strains. Pretreatment of grafted cells with mitomycin C abrogated the PLNE. The treatment of donor cells with anti-Thy or anti-Lyt 1 plus complement but not with anti-Lyt 2 abrogated the reaction, while nylon wool nonadherent splenocytes led to results similar to those obtained with the whole spleen population. These results suggestthat donor Lyt I+ T cells are the cells inducing this reaction. To evaluate the proliferation occurring in the PLN, an in vim-in vitro method (11) was used. Cells from the PLN were harvested and pulsed in vitro with [3H]TdR for measurement of proliferation. The PLN draining the inocula of Lyt l+ splenocytes from reciprocal hybrids showed proliferative levels significantly higher than those induced by splenocytes from identical hybrids, indicating that in situ proliferation accompanied the PLNE observed. Proliferative levels registered were consistently lower than those induced by inoculation of parental splenocytes. When assayswere carried out inoculating T or Lyt l+ splenocytes and treating cells from the draining popliteal lymph nodes with anti-Lyt 1, anti-Lyt 2, or anti-Ig serum plus complement,
FOSTER-NURSING AND PLNE IN SYNGENEIC HOSTS GRAFT
HOST
(PKR x fVWc)Fl
wB/c
183
GRAFT PRETR.
(AKR x WIB/C)~,~F~
x pKR)Fl ml4
(AKR x WLEI/c)Fl
Thy 1.2 /I
(.WR.M/SnxMR)Fl (MR x MR.WSn)Fl
Lyt 1
v'xR.wsn
x PxR)fM II
RATIO
(qmr@tkpnleft)
FIG. 3. [‘H]TdR incorporation by PLN cells from Fl hosts inoculated in the right footpad with 7 X IO6 splenocytes or NWNA cells from foster-nursed Fl hybrids. When indicated, donor cells were treated with the appropriate antibody plus complement. In the contralateral footpad, hosts received the same number of splenocytes from identical Fl hybrids. On Day 3, the PLN nodes were excised, disrupted, and treated with the appropriate antibody plus complement. The obtained cell suspensionswere harvested after an IShr pulse of i3HJTdR. Each bar represents the arithmetic mean of cpm right PLN/cpm left PLN (number of mice, four). SE were lessthan 15%.
in vitro proliferation was significantly decreased,indicating that proliferation of host Lyt 2+ and B cells is involved. It remains unknown whether Lyt l+ cells proliferating on Day 3 are of host or graft origin. Foster-nursing of Fl hybrids on mothers from the paternal strain was able to induce permanent alterations in the ability of splenocytes to induce PLNE: a significant PLNE could be detected when T or Lyt I+ splenocytes from foster-nursed Fl hybrids were grafted in identical normally nursed Fl hosts; on the contrary, no PLNE was detected when splenocytes from foster-nursed F 1 hybrids were inoculated in reciprocal normally nursed Fl hosts. Thus, the pattern of spleen ability to induce PLNE of foster-nursed Fl mice resembled that observed in the reciprocal Fl combination nursed by its own mother. Proliferative events occurring in lymph nodes draining the inoculum of splenocytes from foster-nursed Fl hybrids were found to be similar to those observed with splenocytes from normally nursed reciprocal hybrids. These results suggestthat lactation would be a critical event determining the ability of T splenocytes to induce PLNE when inoculated in identical Fl hosts. When strain combinations between parental strains sharing Mls” antigens were used, neither significant PLNE nor increases in lymph node proliferation could be registered. Foster-nursing of these Fl hybrids did not alter the ability of their splenocytes to induce PLNE. The nature of the alterations reported herein still remains unknown. The induction of qualitative antigenic differences through milk, i.e., the appearance of altered selfcomponents by an endogenous virus cannot be discarded. However, other possibili-
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ties may be considered. The fact that the effect of lactation could be detected in strain combinations involving one parental strain expressing the stimulatory Mls” allele and the other parental strain bearing the nonstimulatory Mlsb allele but could not be detected in those sharing Mls” determinants, raises the possibility that these antigens could be involved. It has been demonstrated that antigenic impacts in newborn mice are able to induce permanent alterations in their immune responsiveness (12-14). Both Mls and Ia antigens seem to be poorly expressed in newborn mice ( 15- 17). On the other hand, it has been recently reported ( 16) that self-reactive (V,‘,) T cells with a mature CD4+ surface phenotype are present during the early postnatal period in the thymus of Mls” mice and that a small portion of these cells can even be detected in the mesenteric lymph nodes of 2- to 3-day-old mice. If, as it has been reported (2), milk cells are able to penetrate in the newborn circulation, the possibility exists that the confrontation between these self-reactive cells and maternal Mls antigens could influence the T cell repertoire of the litter. ACKNOWLEDGMENTS We thank Mr. Juan Portaluppi and Mr. Antonio Morales for efficient technical assistance. This work was made possible by grants from CONICET.
REFERENCES 1. Oksenberg, J. R., Persitz, E., and Brautbar, C., Amer. J. Reprod. Immunol. Microbial. 8, 125, 1985. 2. Weiler, I. J., Hickler, W., and Sprenger, R., Amer. J. Reprod. Immunol. 4,95, 1983. 3. Head, J. R., and Beer, A. E., In “Immunology of Breast Milk” (P. L. Ogra and D. Dayton, Eds.), pp. 207-225. Raven Press,New York, 1979. 4. Nepomnaschy, I., Deroche, A., Pasqualini, C. D., and Piazzon, I., Zmmunol. Lett. 18, 19, 1988. 5. Gazes, Y., Umiel, T., Meshorer, A., and Trainin, N., J. Immunol. 121,2199, 1978. 6. Small, M., and Trainin, N., Cell. Immunol. 20, 1, 1975. 7. Camaud, C., Chaniere, J., and Bach, J. F., Cell. Immunol. 28,274, 1977. 8. L’Age-Stehr, J., and Diamantstein, T., Nature (London) 271,663, 1978. 9. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3,645, 1973. 10. Ford, W. L., Burr, W., and Simonsen, M., Transplantation 10,258, 1970. 11. Chaouat, G., and Chaffaux, S., Amer. J. Reprod. Immunol. 6, 107, 1984. 12. Nosal, G. J. V., Annu. Rev. Immunol. 1,33,1983. 13. Rayboume, R., Arnold, L. W., and Hat&ton, G., J. Immunol. 127, 1142, 1981. 14. Argyris, B. F., Cell. Immunol. 74,313, 1982. 15. Ahmed, A., Scher, I., and Sell, K. W., Cell. Immunol. 30, 122, 1977. 16. Schneider, R., Lees, R. K., Pedrazzini, T., Zinkemagel, R. M., Hengartner, H., and MacDonald, H. R., J. Exp. Med. 169,2149, 1989. 17. Lu, C. Y., Calamai, E. G., and Unanue, E. R., Nature (London) 282,327, 1979.