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Keywords:

  • Ontogeny (comparative immunology);
  • CTL;
  • Tolerance;
  • Graft-vs.-host disease;
  • Transplantation

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Neonatal and adult mice mount distinct responses to allogeneic cells. Injection of neonates with fully allogeneic cells results in lethal graft-vs.-host disease (GVHD), whereas injection of semi-allogeneic (F1) cells leads to lifelong tolerance to the alloantigens, often marked by specific CTL non-responsiveness. In contrast, adults injected with the same number of either cell type become primed and develop vigorous anti-donor CTL activity. One possibility for this differential responsiveness may be developmental immaturity in the CTL arm of the immune system. Recent studies have shown that neonates are capable of mounting mature CTL responses, but only in the presence of strong Th1-promoting agents. Here, we demonstrate that neonates are competent to develop vigorous MHC class I-restricted CTL activity in vivo upon exposure to either fully or semi-allogeneic spleen cells. Specific CTL activity was generated using doses of cells approximately tenfold lower than levels used for the induction of GVHD or tolerance. Thus, the present studies demonstrate that mouse neonates are fully mature in their capacity to develop alloreactive CTL activity, as long as the dose of donor cells is low enough. These results have important implications for the known exposure of human fetuses and infants to small numbers of maternal cells.

Abbreviation:
GVHD:

Graft-vs.-host disease

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The neonatal period has been viewed as a developmental window in which quantitative and/or qualitative immaturity of the immune system results in limited responsiveness. This perspective is atleast partially based on the responses of neonates to alloantigens. The development of alloantigen-specific CTL by neonatal T cells in vitro is poor (reviewed in 1). CTLresponses also appear to be functionally limited in vivo since neonates injected with fully allogeneic cells usually develop a lethal graft-vs.-host disease (GVHD) 2. Neonatal responses to semi-allogeneic (F1) cells also appear to be compromised. Injection of neonates with adult F1 spleen cells results in specific tolerance associated with the lifelong survival of donor F1 skin grafts and anti-donor CTL non-responsiveness (reviewed in 3).

The studies with allogeneic cell transplantation initially suggested that neonates were unable to develop substantial CTL responses. However, a number of laboratories have recently demonstrated that it is possible to elicit mature neonatal CTL responses. In general, CTL responses against viral, bacterial, or parasitic antigens have been obtained using strong Th1-promoting agents. For example, DNA encoding selected proteins 46, combinations of DNA vaccines with DNA encoding GM-CSF 7, CpG-containing oligonucleotides 8, adult DC 9, and exogenous IL-12 10 all promote strong CTL responses in neonates. Thus, it is clear that mouse neonates are competent to develop mature, specific CTL responses under selective circumstances. What is not clear is whether neonates can develop such responses under more natural conditions of exposure, i.e. when challenged in vivo without the aid of strong Th1-promoting agents. This is a particularly important issue for alloantigens. It has been extensively documented 1114 that small numbers of maternal cells are transmitted across the placenta to the fetus or to the newborn via the breast milk, thus exposing the fetus and newborn infant to maternal non-inherited alloantigens.

In an elegant study reported in 1996 9, Matzinger and colleagues demonstrated that adults as well as neonates could be tolerized to minor histocompatibility antigens (H-Y). This was achieved in adults by increasing the dose of donor spleen cells several logs over the dose used to tolerize neonates. These results led the authors to propose that neonates and adults were both susceptible to tolerance induction; "... it simply takes far fewer cells to generate tolerance in a newborn because there are far fewer T cells to turn off." A corollary to this idea is that the introduction of fewer allogeneic cells into the neonate may not result in tolerance but may lead, instead, to the development of mature responses.

In this report, we have directly tested the idea that specific CTL priming may be attainable by lowering the dose of allogeneic cells inoculated. We have reproduced published work showing that neonates indeed become tolerant to the standard number of injected semi-allogeneic cells and die of lethal GVHD using similar numbers of fully allogeneic cells. However, when the injected cell number was reduced approximately tenfold, neonates developed strong and specific CTL responses to both semi- and fully allogeneic cells. Specific CTL activity was generated in neonatal hosts of either H-2b or H-2d genetic background, indicating the universal nature of this capacity. Thus, the specific outcome of injection of allogeneic cells into neonates is highly dependent on the magnitude of the donor cell inoculum; relatively high doses of semi-allogeneic cells elicit tolerance, relatively high numbers of fully allogeneic cells lead to lethal GVHD, whereas lower numbers of either cell type efficiently prime neonates for alloantigen-specific CTL responses.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 Exposure of neonates to low numbers of fully allogeneic spleen cells does not lead to tolerance

Experiments to study GVHD induced by the injection of BALB/c (H-2d) spleen cells into C3H.SW (H-2b) neonates indicated that survival improved as the number of cells injected was decreased. Notably, at a dose of 2×106 cells, approximately tenfold lower than our "standard" dose of 2×107 cells, 100% of the animals survived, without showing any of the typical clinical signs of GVHD (data not shown; see below). One possible explanation for this phenomenon was that the lowered dose of donor cells was inducing tolerance in the neonates. A hallmark of neonatal tolerance is that skin grafts of donor type will not be rejected, even when the host animals have grown to adulthood. Therefore, skin graft analysis was used to test for potential tolerance induction in this system. C3H.SW neonates were injected with 2×106 BALB/c spleen cells; a parallel group of uninjected C3H.SW neonates were used for control mice. Seven weeks later, the animals were grafted with skin from BALB/c (donor) and C3H.HeJ (H-2k; third party) adult mice. All mice rejected third-party and donor skin grafts; similar kinetics of rejection was observed for the control and the BALB/c spleen cell-injected mice (Fig. 1). Thus, low doses of fully allogeneic cells did not appear to induce tolerance in neonates.

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Figure 1. Neonates injected with small numbers of fully allogeneic spleen cells readily reject donor skin grafts. C3H.SW mice (1-day-old) were injected with 2×106 spleen cells from adult BALB/c mice. A parallel set of aged-matched mice were maintained as uninjected control animals. Seven weeks later, all mice were grafted with skin from adult BALB/ c (donor) or C3H/HeJ (third party) mice. The grafts were then examined daily for signs of rejection. Control syngeneic C3H.SW grafts looked healthy out to 20 days post-grafting (data not shown). Each symbol represents an individual animal. One-way ANOVA analysis indicated that there was no statistical differences between groups (p=0.2791).

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2.2 Long-term priming for donor-specific cytotoxic responses in neonates injected with small numbers of fully allogeneic cells

In initial experiments, we found that donor cells were present in the spleens of C3H.SW neonates 2–4 days following the injection of 2×106 BALB/c spleen cells; however, by 7 days post-injection, donor cells were no longer detectable (J. Richardson and R. B. Levy, data not shown). Because donor cells were present early after administration, their ultimate disappearance seemed unlikely to be due to rapid, nonspecific removal mechanisms (e.g. trapping and destruction in the lungs or liver). Rather, the kinetics of elimination was suggestive of the potential development of primary T cell responses, most likely host anti-donor cytotoxicity. To test this hypothesis, C3H.SW neonates were administered 2×106 BALB/c spleen cells; uninjected age-matched C3H.SW neonates were maintained as control animals. Three weeks following injection, spleens were removed and pooled (Fig. 2). One pool from uninjected control animals (three mice) and two pools (P1, P2; three mice/pool) from a total of six injected neonates were made. Responding cells were stimulated with BALB/c (H-2d, donor) irradiated spleen cells for 5 days and cytotoxic activity against donor-, host-, or third party-type cells was tested in a standard 4-h 51Cr-release assay.

As expected, all animals (uninjected and injected) demonstrated minimal reactivity against MHC ‘self’ H-2b matched (EL4) target cells (Fig. 2). In contrast, lysis of 51Cr-labeled P815 (H-2d, donor-type) target cells was observed by effector cells from both uninjected control and injected animals (Fig. 2) at high E/T ratios (40:1). However, lysis by cells from uninjected control cells was clearly weaker, declining rapidly at lower E/T ratios, while that of injected animals remained high (>70% specific lysis at an E/T ratio of 5:1). Prior stimulation was required since there was no detectable lysis with cells cultured in the absence of stimulation (data not shown). Together, the specificity of the response and the high level of lysis (70–90%) against donor H-2d target cells indicate that C3H.SW neonates generated a primed cytotoxic response following injection with fully allogeneic BALB/c cells.

To determine whether this priming effect was long lasting, additional groups of animals were similarly analyzed 6 weeks following exposure to alloantigen. In these experiments, allospecific responses that develop in vitro among cells that were not primed in vivo were minimized by limiting the stimulation period to 3 days. Again the injected neonates (as well as adult injected mice) restimulated with BALB/c antigen demonstrated lysis of the donor-type (51Cr-labeled P815) target cell population (Fig. 3). Effector cells from all animals exhibited minimal lysis of either 51Cr-labeled H-2b (host-type) or 51Cr-labeled H-2k (third party) target cells (Fig. 3). Thus, long-term priming for donor-specific cytotoxic responses can be induced in mice injected with allogeneic cells at 1 day of age.

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Figure 2. Donor-specific cytotoxicity develops within 3 weeks of injection of 1-day-old C3H.SW neonates with small numbers of fully allogeneic BALB/c spleen cells. C3H.SW neonates (1-day-old) were injected with 2×106 spleen cells from adult donor BALB/c mice. Uninjected age-matched C3H.SW neonates were maintained as control animals. Three weeks after injection, spleens were harvested. Cells from uninjected mice (squares; n=3) were pooled and two pools [pool 1 (circles), pool 2 (triangles)] from injected animals (n=3/pool) were produced. Cells from each pool were stimulated for 5 days in vitro with irradiated donor (BALB/c, H-2d) splenocytes. Cytotoxic killing was assessed in a 4-h 51Cr-release assay against donor-type (P815, H-2d), host-type (EL4, H-2b), and third-party (BW5147, H-2k) target cell populations, as indicated. The percent specific lysis at the indicated E/T ratios is presented. Lytic units: pool 1: 2,485.3; pool 2: 1,258.6; uninjected: 332.7.

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Figure 3. Neonates injected with fully allogeneic cells exhibit primed donor-specific cytotoxic responses 6 weeks post-injection. C3H.SW neonates (1-day-old) or adults were injected with, respectively, 2×106 or 2×107 spleen cells from adult donor BALB/c mice. Uninjected age-matched C3H.SW neonates were maintained as control animals. Six weeks after injection, spleen cells were harvested and randomly pooled (n=3/group) as described in Fig. 2. Spleen cells from each group were stimulated for 3 days in vitro with irradiated donor (BALB/c, H-2d) splenocytes. Cytotoxic killing was assessed as described for Fig. 2. Lytic units: pool 1: 1,190.7; pool 2: 950.9; uninjected: 495.7; adult injected: 990.9.

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2.3 Anti-donor cytotoxicity is mediated by CD8+ cells

Cytotoxicity can be mediated by a number of cell types. To determine whether we were observing a CD8+ CTL response, cytotoxicity assays were conducted in the presence or absence of anti-MHC class I mAb directed against donor-type target cell MHC molecules (Fig. 4). Specific lysis of donor-type targets by effector cells from 3-week-old C3H.SW mice was inhibited in the presence of anti-donor MHC class I mAb (Fig. 4). Cytotoxicity by adult C3H.SW CTL was also reduced in the presence of these antibodies. This result, together with the lack of detection of cytotoxicity against NK sensitive targets (YAC-1, data not shown), supports the notion that injection of 1-day-old neonates with fully allogeneic cells primes CD8+ T cells for anti-donor CTL activity.

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Figure 4. Neonatally primed cytotoxic responses are directed against donor MHC class I molecules. C3H.SW neonates or adults were injected with adult BALB/c cells as described in Fig. 3. Three weeks after injection, spleen cells were stimulated for 3 days in vitro with irradiated donor-type (BALB/c, H-2d) splenocytes. Target cells (P815, H-2d) were pre-incubated for 10 min at room temperature in micro-wells containing media alone (open symbols) or mAb against MHC class I Kb, Dd, Ld (filled symbols) prior to the addition of effector cells. Lytic units: uninjected: 20.4 (no mAb) vs. 17.3 (plus mAb); injected pool: 114.6 (no mAb) vs. 25.4 (plus mAb); adult injected: 162.1 (no mAb) vs. 56.4 (plus mAb).

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2.4 Priming of neonates to fully allogeneic cells is universally observed among different mouse strains

These experiments were conducted using C3H.SW (H-2b) mice as hosts and BALB/c (H-2d) animals as donors. H-2b-genotype mice typically generate strong cell-mediated immune responses (reviewed in 15, 16) and, thus, would be expected to develop vigorous CTL responses. To determine whether the phenomenon we were observing was limited to H-2b-genotype hosts or perhaps exclusively to the C3H.SW strain of mice, the donor and host strains were reversed. BALB/c mice are prototypically a Th2-biased strain (reviewed in 15, 16). Thus, using BALB/c neonates as hosts would simultaneously test whether primed CTL responses could be generated in other host genotypes and whether a more Th2-biased strain was competent to mount these responses.

In an initial experiment, 1-day-old BALB/c neonates were administered 2×106 adult C3H.SW splenocytes, i.e. the same dose of cells that elicited anti-BALB/c responses in C3H.SW neonates. Over the next 2–3 weeks, these BALB/c mice became moribund, exhibiting symptoms of hunched posture, limited mobility, ruffled fur, and diarrhea, and many subsequently died (data not shown). Since these symptoms are consistent with the development of GVHD, we reasoned that the "switch" from GVHD to specific CTL priming might occur at a lower dose of cells when using this donor/host combination.

To investigate this possibility, BALB/c neonates were exposed to varying numbers of adult C3H.SW spleen cells and viability was monitored (Fig. 5). All BALB/c neonates injected with 4×106 C3H.SW spleen cells died by 22 days post-injection (Fig. 5A). Similar to the initial find-ings, a dose of 2×106 C3H.SW spleen cells resulted in approximately 50% mortality. However, at 1×106 cells, 80% of the host neonates survived, and at a dose of 5×105 cells, i.e. a dose fourfold lower than that used in the reverse donor/host combination, 100% of the BALB/ c neonates survived. Notably, among the surviving mice at 3 weeks post-injection of 5×105, 1×106, or 2×106 cells, all groups exhibited clear anti-donor CTL activity (Fig. 5B).

BALB/c neonates were then exposed to a different H-2b, i.e. C57BL/6, spleen cell inoculum. The survival results obtained with decreasing C57BL/6 donor cell numbers were similar to the findings following injection with C3H.SW cells (Fig. 6A). Nine to ten weeks following injection of donor C56BL/6 cells, CTL priming in these BALB/c mice was also detected (Fig. 6B). Thus, the development of CTL priming in neonates to fully allogeneic cells is not limited to the C3H.SW strain or to host strains of the H-2b genotype. Moreover, this priming appears to be stably maintained and is readily detectable in mice at 9–10 weeks of age.

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Figure 5. BALB/c neonates injected with decreasing numbers of C3H.SW spleen cells generate primed anti-donor cytotoxic responses. (A) BALB/c neonates (1 day old) were injected with the indicated numbers of adult C3H.SW spleen cells. Survival was monitored daily out to 22 days post-injection. Differences in survival time between groups was statistically significant (p<0.0001) as determined by one-way ANOVA analysis. (B) Three weeks post-injection, spleen cells were stimulated for 3 days in vitro with donor-type cells (C3H.SW, H-2b). Cytotoxic killing was assessed against H-2b (EL4) target cell populations. Lytic units: 2×106: 102.9; 1×106: 193.8; 5×105: 130.2; uninjected: 26.0; adult injected: 465.5.

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Figure 6. CTL priming is also induced in BALB/c neonates injected with C57BL/6 (H-2b) spleen cells. (A) BALB/c neonates (1-day-old) were injected with the indicated numbers of adult C57BL/6 (B6) spleen cells. Survival was monitored daily out to 22 days post-injection. Differences in survival time between groups were statistically significant (p=0.0017) as determined by one-way ANOVA analysis. (B) Nine to ten weeks after injection, spleen cells were stimulated with donor-type cells (C57BL/6, H-2b) in vitro for 3 days. Cytotoxic killing was assessed against H-2b (EL4) target cell populations. Lytic units: 5×105: 101.4; uninjected: 9.5.

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2.5 Specific priming of neonates to fully allogeneic cells requires donor CD4+ cells

Under most conditions of antigen exposure, adaptive immune responses in neonates are blunted (reviewed in 1719). Thus, we hypothesized that one or more of the mature adult cell types in the donor inoculum may be responsible for "helping" neonates to develop CTL function. To test this idea, BALB/c neonates were injected with spleen cells from CD4- or CD8-deficient mice. The two positive control groups consisted of neonates injected with the same number of (a) spleen cells from wild-type C56BL/6 mice and (b) a mixture of spleen cells (1:1) from CD4- or CD8-deficient mice. A cohort of neonates was also maintained as the negative control, uninjected group. Nine weeks later, spleen cells were stimulated with donor-type (C57BL/6, H-2b) irradiated splenocytes and tested 3 days later for cytotoxic activity against donor-type cells (Fig. 7).

As expected, the uninjected negative control group showed minimal lysis at all E/T ratios. Cells from neonates injected with wild-type cells, with CD8-deficient cells, or with a mixture of CD4- and CD8-deficient cells all demonstrated anti-donor cytotoxic activity. In contrast, cells from neonates injected with CD4-deficient cells showed no to low lytic activity. These experiments indicate that the presence of donor CD4+ cells is necessary for the development of neonatal CTL function upon exposure to fully allogeneic cells.

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Figure 7. Donor CD4+ cells are required for priming against fully allogeneic cells. BALB/c neonates were injected with 5×105 spleen cells from wild-type mice (filled circles) or from CD4- (filled squares) or CD8- (filled triangles) deficient mice. One group of neonates also received a mixture (1:1) of cells from CD4-or CD8- deficient mice (filled triangles) and a final group was maintained as uninjected control animals (open circles). Nine weeks later, spleen cells were stimulated with donor-type cells (C57BL/6, H-2b) in vitro for 3 days. Cytotoxic killing was assessed against H-2b (EL4) target cell populations. Lytic units: wild-type: 92.4; CD4–/–: 17.8; CD8–/–: 136.9; CD4–/– + CD8–/–: 64.2; uninjected: 32.7.

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2.6 Priming of neonatal CTL responses can also be achieved at low doses of semi-allogeneic cells

Unlike fully allogeneic cells that cause GVHD in neonates, the "standard" injected dose of semi-allogeneic cells induces specific tolerance in neonates. The results described above indicated that it was possible to achieve specific CTL priming, rather than GVHD, as long as low enough numbers of fully allogeneic donor cells were introduced. These results prompted us to ask whether CTL priming, rather than tolerance, could be generated in neonates using lower numbers of semi-allogeneic donor cells. To address this question, C3H.SW neonates were administered different numbers of spleen cells from (C3H.SW×BALB/c) F1 adult donors. In parallel, one group of C3H.SW neonates was maintained as uninjected controls and a third group was injected with 2×106 fully allogeneic BALB/c spleen cells. Three weeks after injection, spleen cells were stimulated with donor-type (BALB/c, H-2d) irradiated splenocytes.

At all E/T ratios, there was minimal to no lysis of the targets by cells from uninjected control animals or from animals that had received 1×107 or 2×107 F1 cells (Fig. 7). Since this is the dose of cells commonly used to achieve tolerance to semi-allogeneic donor cells, poor lysis is probably indicative of specific tolerance. This idea is underscored by the observation that the same responding cells developed CTL activity when stimulated with third-party cells in parallel cultures (data not shown). Cells from animals that received 5×106 F1 donor cells showed specific lysis about 1.5-fold higher than that of cells from control animals. Notably, animals that received the lowest dose (2×106) of F1 cells showed the highest level of anti-H-2d specific lysis and these responses were comparable to those that developed in neonates receiving a similar number of fully allogeneic cells (Fig. 8). A primed, rather than a tolerized response in this group of mice to the donor cells was supported by their efficient rejection of donor skin grafts (data not shown). Thus, when neonates encounter low doses of semi-allogeneic F1 cells, efficient CTL priming, rather than tolerance, develops.

To determine whether complete donor/host MHC differences were required for the development of CTL priming in neonates, H-2Kb mutant spleen cells were used as donors. Effectors from bm1-injected neonates 3 and 6 weeks later showed substantial lysis of bm1 target cells (data not shown). In total, these results indicate that efficient CTL priming can be achieved in neonates when fully or semi-allogeneic donor spleen cells are administered.

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Figure 8. Specific priming for CTL activity in neonates injected with low numbers of semi-allogeneic cells. C3H.SW neonates (1-day-old) mice were injected with the indicated numbers of spleen cells from adult (BALB/c × C3H.SW) F1 or BALB/c mice. Uninjected age-matched C3H.SW neonates were maintained as control animals (no cells). Six weeks after injection, spleen cells from each group of animals (n=3–4/group) were stimulated with donor-type (BALB/c, H-2d) cells in vitro for 3 days. Cytotoxic killing was assessed against H-2d (P815) target cell populations. Lytic units: uninjected: 33.9; 2×107 F1: 3.3; 1×107 F1: 12.0; 5×106 F1: 56.8; 2×106 F1: 152.6; 2×106 BALB/c: 152.5.

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Recently, the in vivo development of CTL responses by murine neonates has been reported 410. In general, these activities were elicited in the presence of strong Th1-promoting agents. These results are of clear importance since they demonstrate that neonates are fully mature in their capacity to develop cytotoxic function, at least under selected conditions of antigen presentation. However, whether neonates were similarly competent to develop CTL responses in the absence of strong Th1-driving substances remained unclear. The present studies have addressed this issue by re-investigating the capacity of neonates to develop alloantigen-specific cytotoxic responses in vivo. We demonstrate that the responses of mouse neonates to allogeneic spleen cells are highly dependent on the numbers of cells administered. The crucial nature of these cell numbers was manifest by a ‘shift’ in the response in neonates from (a) GVHD to priming using fully allogeneic spleen cells and (b) tolerance to priming using semi-allogeneic spleen cells.

The "standard" high dose (2×107 i.v.) of fully allogeneic cells produced a lethal GVHD in neonates, as expected 2. However, reduction of the number of cells approximately tenfold resulted in efficient alloantigen priming for cytotoxicity. Anti-donor specific CTL responses were elicited in both H-2b- and H-2d-strain neonates and skin grafts of donor type were readily rejected. Moreover, the induction of neonatal anti-donor cytotoxicity was dependent on the presence of CD4+ cells in the donor inoculum. For semi-allogeneic F1cells, the "standard" high dose resulted in specific tolerance, again as predicted 20. Similar to the experiments with fully allogeneic cells, reduction of the injected cell number approximately tenfold led to priming of donor-specific CTL responses. Thus, these findings demonstrate that neonates are fully competent to develop mature, donor-specific CTL responses when exposed to relatively low numbers of either semi- or fully allogeneic spleen cells.

The present results have major implications for exposure to alloantigens in early life. As originally appreciated first in the 1960's, fetuses and newborns are exposed to non-inherited maternal antigens by transmission of maternal cells across the placenta or in the breast milk 21, 22. The numbers of transmitted maternal cells are small and their detection has recently been facilitated with sensitive molecular techniques, such as PCR or in situ hybridization 2327. Observations in human transplantation support the idea that this maternal cell transmission has a lifelong effect on the ability of the offspring to recognize alloantigen. There is evidence that tolerance to maternal alloantigens may occur following early life exposure 28. However, a higher incidence of early allograft rejection was observed in recipients of kidneys from siblings expressing maternal HLA antigens not inherited by the recipient than in recipients of kidneys from siblings expressing paternal HLA antigens not inherited by the recipient 29. Thus, fetal and neonatal exposure tomaternal antigens in humans may, in some instances, lead to immunological priming.

Our results provide a cellular mechanism that could account for these observations, i.e. whether tolerance or priming is achieved in early life appears to be highly dependent on the exposure dose. The "switch" from one response to the other occurs in mouse neonates over a fairly narrow range (2–4×) of cell numbers. Thus, it is tempting to speculate that, in humans, the different outcomes seen following transplantation may be a reflection of fetal or neonatal exposure to different numbers and/or types of maternal cells.

A particularly striking finding in this study is that, in the absence of donor CD4+ cells, the development of cytotoxicity against fully allogeneic cells is essentially eliminated (Fig. 7). This result indicates that donor CD4+ cells are necessary for neonates to develop CTL alloreactivity. Although other explanations are possible, this phenomenon might be due to the provision of mature CD40L signals by the donor adult CD4+ cells. There is evidence that mouse neonatal T cells poorly up-regulate CD40L 30. The CD40/CD40L interaction induces the production of the Th1-promoting cytokine IL-12 by APC (reviewed in 31) and has been shown to be one of the critical costimulatory pathways for the full activation of T cells during alloimmune responses 3136. Thus, by providing mature CD40L expression, the donor adult CD4+ cells may promote the development of host alloreactive CTL. A clear prediction from this idea is that the development of neonatal CTL responses will be compromised when donor cells are from fully allogeneic CD40L-deficient animals. Experiments to test this proposal are currently underway.

The cellular interactions leading to neonatal CTL reactive with semi-allogeneic cells are currently unknown. In this case, it is unlikely that donor T cells are involved in the promotion of host CTL responses since the F1 donor cells are tolerant to the neonatal host antigens. One intriguing possibility involves host regulatory T cell function. CD8+ regulatory cells have been implicated in down-modulating neonatal Th2-mediated pathologies in the mouse 37 and in the rat 38. On the other hand, CD4+ regulatory cells may play a role in staunching host anti-donor Th1/CTL activity when neonates are injected with high numbers of semi-allogeneic cells 3, 39. Lowering the number of semi-allogeneic cells, as in our system, may then favor the activation of CD8+ over CD4+ regulatory cells. The net result could be the preferred promotion of Th1 and CTL function under these circumstances. Again, there is a clear prediction to this hypothesis: neonates injected with high numbers of semi-allogeneic cells will develop CD4+ regulatory cells with the capability of down-regulating anti-donor CTL responses 3, 39, while neonates injected with low numbers will not. Thus, our system provides a unique opportunity to learn about development of the Th/CTL arms of immunity and the regulatory processes governing their function.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Mice

BALB/c mice were originally obtained from Charles River Laboratories (Wilmington, MA); C3H.SW and C3H/HeJ mice were from The Jackson Laboratory (Bar Harbor, ME); CD4- or CD8-deficient mice on a C57BL/6 background were also purchased from The Jackson Laboratory. Animals were bred and housed under barrier conditions in the Division of Veterinary Resources at the University of Miami MedicalSchool. Day of birth was called day 0.

4.2 Exposure of neonatal animals to alloantigen

Total spleen cells were depleted of red cells and washed twice with Hanks' balanced salt solution prior to injection. One-day-old neonates or adult mice were injected with the indicated number of spleen cells; neonates received a total volume of 50 μl in the facial vein, adults were injected with a total volume of 250 μl in the tail vein.

4.3 Medium and culture reagents

Complete medium utilized for all cultures consisted of RPMI 1640 containing 1 mM sodium pyruvate, 2 mM L-glutamine, 5×10-2 mM 2-ME, 1% nonessential amino acids, 1% penicillin-streptomycin, and 10% FCS. All medium components were purchased from Life Technologies (Grand Island, NY).

Con A supernatant was obtained from 48-h cultures of rat spleen cells (5×106/ml) stimulated with Con A (5 μg/ml). Residual Con A was removed by adding 0.2 M α-methyl-mannoside (Sigma Chemical Co., St. Louis, MO).

4.4 Cell-mediated cytotoxicity assay

The induction of cytotoxic T cell activity has been described previously 40. In brief, 6×106 spleen cells were cultured in vitro with 3×106 irradiated (2.0 Gy) allogeneic spleen cells for 3-5 days at 37°C in 24-well plates. Effector cells generated were assayed on 51Cr-labeled stimulated target cells (2×106/ml spleen and lymph node cells plus 5.0 μg/ml Con A for 48–72 h at 37°C) or the following tumor cell lines: EL4 (H-2b), P815 (H-2d), BW5147 (H-2k). Percent lysis and specificlysis were calculated as described above. Specific lysis is presented as the mean value of triplicate cultures. The error of the mean was consistently between 2% and 6%. Lytic units of cell-mediated cytotoxicity were calculated according to a formula previously reported 41. Calculations are based on utilization of an E/T ratio which gives 20% lysis and values represent lytic units/1×107 effector cells.

4.5 Skin grafting

The procedures for skin grafting have been described in detail previously 42. Briefly, donor skin was placed on a prepared site on the host tails and protective plastic tubes with air holes were placed over the tails. The tubes were removed on day 2–3 after grafting. Beginning on days 4-5 after grafting, the conditions of the grafts were assessed with the aid of a dissecting microscope. Complete rejection was defined as shriveling, followed by drying and flaking away of the graft.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

This study was supported by NIH grants: RO1 AI44923–02 (B. Adkins); R01 AI46689 and R01 RR11576 (R. B. Levy).

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    WILEY-VCH

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    WILEY-VCH

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    WILEY-VCH

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    WILEY-VCH

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    WILEY-VCH

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    WILEY-VCH

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    WILEY-VCH

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    WILEY-VCH

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