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. 2008 Nov;125(3):387–396. doi: 10.1111/j.1365-2567.2008.02855.x

Susceptibility of neonatal T cells and adult thymocytes to peripheral tolerance to allogeneic stimuli

Fábio B do Canto 1, Celso Lima Junior 1, Ivan A Teixeira 1, Maria Bellio 2, Alberto Nóbrega 2, Rita Fucs 1
PMCID: PMC2669142  PMID: 18462348

Abstract

We studied the tolerization of neonatal thymocytes (NT), neonatal splenocytes (NS) and adult thymocytes (AT), transferred to syngeneic nude (nu/nu) hosts previously injected with semi-allogeneic splenocytes, without any supportive immunosuppressive treatment. This protocol allows the study of peripheral tolerance in the absence of the thymus. BALB/c neonatal T cells and ATs were able to expand in syngeneic BALB/c nu/nu mice and functionally reconstituted an allogeneic response, rejecting (BALB/c × B6.Ba) F1 splenocytes transferred 3–4 weeks after injection of BALB/c cells. However, if (BALB/c × B6.Ba) F1 cells were injected into BALB/c nude hosts 30 days before transfer of NT, NS or AT cells, the F1 population was preserved and specific tolerance to B6 allografts was established. Furthermore, transfer to lymphopenic F1 nu/nu showed that tolerance could be established only for neonatal populations, showing that unique properties of neonatal T cells allow their tolerization in both lymphopenic and non-lymphopenic conditions, in the absence of suppressive immunotherapy. These results bring empirical support to the possibility of T-cell engraftment in immunodeficient patients showing partial identity with donor major histocompatibility complex (MHC) genes; the manipulation of immunological maturity of donor T cells may be the key for successful reconstitution of immunocompetence without induction of graft-versus-host disease.

Keywords: alloreactivity, neonatal tolerance, peripheral tolerance, recent thymus emigrants, regulatory T cells

Introduction

The particular receptiveness of the neonatal period to tolerance induction was established a long time ago, when the classical Medawar protocol was developed.1 Although the mechanisms involved in this tolerance process have not been elucidated, the general notion of ‘functional immaturity’ of the neonatal immune system has been put forward to account for the absence of the immediate immune rejection of F1 splenocytes by the neonatal parental-type host. According to this hypothesis, stimulation of neonatal T cells by semi-allogeneic antigen-presenting cells (APCs) would anergize or lead to apoptosis of the alloreactive neonatal lymphocytes, allowing the long-term persistence of chimerism, which is thought to be responsible for clonal deletion of newly formed alloreactive thymocytes.

More recently, the contribution of regulatory T cells (Tregs) to the tolerance obtained with the Medawar protocol was suggested.2,3 The peculiar properties of the neonatal thymic microenvironment have been associated with tolerance acquisition4–12 and may be particularly favourable for thymic Treg selection and action. However, it is not known if the entry of donor F1 cells into the thymus is required for Treg formation or if post-thymic interactions may also lead to the generation of Tregs in the periphery. The lymphopenic peripheral compartment of the neonate, which allows vigorous expansion of the first T cells to emigrate,13,14 may also allow an increased rate of conversion of naïve CD25− to regulatory CD25+ T cells in the periphery.15 Also, the recent thymus emigrant (RTE) nature of the peripheral T cells in neonates may be important to permit the action of Tregs, as RTE lymphocytes are more easily recruited to a regulatory phenotype.16,17

In adult animals, transplantation tolerance induction across the major histocompatibility complex (MHC) barrier is notoriously difficult to establish in the absence of immunosupression and is strongly associated with the presence of Tregs. A few protocols were successful in obtaining long-term tolerance to allo-MHC, achieving the post-thymic conversion of adult naïve T cells to a regulatory phenotype.18–22 These include the use of blocking antibodies to T-cell activation molecules18,23 or T-cell expansion in lymphopenic conditions.15 We have recently designed a new protocol that allows long-term allo-tolerance to be obtained without immunosuppressive treatment, using enriched ratios of Treg:T naïve cells together with linked presentation of the allogeneic and syngeneic epitopes on semi-allogeneic F1 APCs.24 This new model system consists of adult BALB/c nude (nu/nu) mice reconstituted with adult (BALB/c × B6) F1 splenocytes and further injected, 1 month later, with adult BALB/c splenocytes enriched for Tregs. The time interval between cell transfers allows F1 T cells to proliferate and reconstitute the T-cell compartment of the recipient nu/nu mouse before injection of the BALB/c cells. In this non-lymphopenic system, the BALB/c splenocytes enriched for Tregs do not reject F1 cells, and the host exhibits tolerance to both B6 and BALB/c skin grafts.

In the present study, we used this F1-reconstituted BALB/c nu/nu model system to compare the acquisition of peripheral tolerance in the absence of the thymus by different T-cell populations [neonatal thymocytes (NTs)/splenocytes (NSs) or adult thymocytes (ATs)]. The results obtained show that NTs/NSs or ATs can acquire allo-tolerance in the absence of the thymus without the need to enrich for CD25+ Tregs, in contrast with adult splenic T cells.24 Interestingly, our data reveal a correlation between tolerance and absence of long-term engraftment of donor lymphocytes. Furthermore, using an analogous protocol of cell transfer into (BALB/c × B6) F1 nu/nu lymphopenic hosts, we show that, under these conditions, only NTs or NSs can be tolerized, revealing a unique property of these populations, which confers immunocompetence without the induction of graft-versus-host disease (GVHD).

Materials and methods

Mice

BALB/c nu/+ (BALB/c), C57BL/6.Thy1.1+ (B6.Ba), (BALB/c nu/+ × B6.Ba) F1 (F1), and CBA/J (H-2k) mice, bred in our conventional animal house (NAL/Universidade Federal Fluminense, Niterói, Brazil), were used as donors of cells suspensions and/or tail skin for graft rejection tests. B6.Ba are homozygous for the Thy1.1+ allele. BALB/c nu/nu (H-2d) mice and (C57BL/6 × BALB/c) F1 nu/nu mice (F1 nu/nu), used as hosts, were housed in micro-isolator cages and received sterilized food and water. The Ethics Committee for Animal Experimentation of the Universidade Federal Fluminense approved the experimental protocols.

Cell transfers

Thymus or spleen cell suspensions were diluted in phosphate-buffered saline (PBS) after red cell lysis by ammonium chloride and counted in the presence of trypan blue. Mice 1–7 days old were used as donors of neonatal populations and mice 30–90 days old were used as adult donors. All cell suspensions were injected intravenously into 20- to 30-day-old BALB/c nu/nu or F1 nu/nu recipient mice. In some transfers, cells were labelled with carboxi-fluorescein succinymidyl ester (CFSE)-5 μM. The cell numbers injected in each experiment and the time schedule are indicated in the figure legends.

Fluorescence-activated cell sorter (FACS) analysis

Immunofluorescent staining and analysis of peripheral blood leucocytes, spleen, lymph node or peritoneal fluid cell suspensions were performed by flow cytometry (FACScalibur; Becton & Dickinson, Franklin Lakes, NJ), using the monoclonal antibodies anti-Thy-1.1-fluorescein isothiocyanate (FITC) (MRC OX-7; Serotec, Oxford, UK), anti-Thy-1.2-phycoerythrin (PE) (30-H12; Gibco, Carlsbad, CA), anti-H-2Kd-biotin (SF1-1.1; BD Pharmingen, San Jose, CA), anti-H-2Db-FITC (CTDb, Serotec) anti-CD4-allophycocyanin (H129.19; Pharmingen), anti-CD25-biotin (PC61, Pharmingen) and anti-forkhead box P3 (Foxp3)-PE (FJK-16s; eBioscience, San Diego, CA). Avidin-FITC (Sigma 083H4825; St Louis, MO) or Avidin-PE Cy5.5 (eBioscience) was used in conjunction with biotin-labelled monoclonal antibodies. Cell membrane permeabilization in Foxp3 stainings was performed by treatment with saponin 0·1%. Paraformaldeid 2% solution was used for cell fixation.

Skin grafting

Full-thickness tail skins from female BALB/c, B6 and CBA/J mice were grafted onto the dorsum of each experimental nude mouse. Grafts were inspected three times per week for hair growth and integrity of the grafted skin (acceptance) or were considered to be rejected when total absence of hair growth together with numerous disruption in tissue integrity, leaving < 20% of the original graft, was noted.

GVHD scores

The following clinical signs of GVHD were noted: (i) reduction of body weight; (ii) protracted posture; (iii) dermatitis; (iv) chronic diarrhoea often leading to mortality.

Results

Tolerance to semi-allogeneic stimuli in chimeric BALB/c nu/nu mice injected with adult (BALB/c × B6.Ba) F1

Tolerance to semi-allogeneic stimuli was studied using the model system previously described, consisting of adult BALB/c nu/nu mice injected with adult (BALB/c × B6.Ba) F1 splenocytes; in this system F1 T cells proliferate and significantly reconstitute the T-cell compartment of the recipient nu/nu mouse, which acquires immunocompetence to mount immune responses and reject third-party allografts. If these [(BALB/c × B6.Ba) F1 → BALB/c nu/nu] animals are further injected 30 days later with adult BALB/c splenocytes, the semi-allogeneic F1 cells are eliminated by mature T cells of the BALB/c donor (Fig. 1a, AS plot); in contrast, the injection of ATs does not lead to the elimination of F1 cells (Fig. 1a, AT plot). Full persistence of F1 cells was also observed after injection of BALB/c NTs or NSs (Fig. 1a, NT and NS plots). A compilation of data from several experiments, showing the percentage of F1 cells in nude hosts, is presented in Fig. 1(b). All animals injected with NTs accepted B6 skin grafts indefinitely and rejected third-party CBA skin grafts with normal kinetics (Fig. 2).

Figure 1.

Figure 1

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Neonatal thymocytes/splenocytes and adult thymocytes acquire allogeneic tolerance when they interact with semi-allogeneic F1 cells in the periphery. (a) Flow cytometry analysis on day 60 of the persistence of F1 (Thy-1.1+/Thy-1.2+) cells in the blood of BALB/c nude (nu/nu) mice receiving adult F1 splenocytes (3 × 106) on day 0 and BALB/c cells of the following populations on day 30: NT, 30 × 106 neonatal thymocytes (n = 10); NS, 30 × 106 neonatal splenocytes (n = 10); AT, 20 × 106 adult thymocytes (n = 13); AS, 20 × 106 adult splenocytes (n = 15); the dot-plots are representative of the data obtained in the different groups. (b) Compiled data from all animals referred in (a), showing percentage of Thy1.1+/Thy1.2+. (c) Flow cytometry analysis on day 60 of the persistence of F1 (Thy-1.1+/Thy-1.2+) cells in the blood of BALB/c nu/nu mice receiving: (NT) 30 × 106 BALB/c neonatal thymocytes (n = 4) on day 0 and 30 × 106 F1 cells on day 30, or (NS) 30 × 106 neonatal splenocytes (n = 7) on day 0 and 30 × 106 F1 cells on day 30; the dot-plots are representative of the data obtained in the different groups. (d) Compiled data from all animals referred in (c), showing percentage of Thy1.1+/Thy1.2+.

Figure 2.

Figure 2

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Neonatal thymocyte (NT) tolerance to alloantigens by peripheral interaction with F1 spleen cells also results in acceptance of allogeneic skin grafts. BALB/c nude (nu/nu) mice previously injected (day 0) with F1 spleen cells (30 × 106) and 1 month later (day 30) with BALB/c NTs (20 × 106) received skin grafts at day 60 from BALB/c, B6 and CBA mice and were analysed for the kinetics of graft rejection.

In most cases where F1 cells were not eliminated after injection of AT, NS or NT BALB/c cells, BALB/c populations did not persist in the host; persistence of BALB/c was observed in 15–20% of the analysed mice. Flow-cytometric analyses of T cells from different compartments of the nude hosts (blood, peritoneal cavity, spleen and lymph nodes) are shown in Fig. 3: in a few cases, BALB/c T cells expanded and persisted and the animals became chimeric, harbouring both the F1 and the BALB/c lymphocytes (Fig. 3a); most animals, however, retained only the F1 cells, as shown in Fig. 3(b); in the rare case where F1 cells were displaced in the AT-injected host (Fig. 3c), the BALB/c lymphocytes expanded and persisted. It is important to note that, although BALB/c ATs do not persist in most cases, they initially proliferate when transferred to BALB/c nu/nu hosts previously reconstituted with F1 splenocytes, as verified by carboxi-fluorescein succinymidyl ester (CFSE) dilution of the injected T cells (Fig. 3d); for comparison, proliferation of BALB/c ATs injected into syngeneic mice [BALB/c → BALB/c nu/nu] is also shown (Fig. 3e).

Figure 3.

Figure 3

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Persistence of F1 splenocytes does not always result in chimerism with BALB/c transferred cells. The presence of F1 (double Thy-1.2+/H-2Db+) cells and of BALB/c (single Thy-1.2+/H-2Db–) cells was determined by flow cytometry on day 60 after transfer, in blood and different peripheral lymphoid compartments [peritoneal cavity (PC), spleen and lymph nodes (LN)] of BALB/c nude (nu/nu) mice into which F1 splenocytes (30 × 106) were transferred on day 0 and adult BALB/c thymocytes (20 × 106) on day 30. The results for the two animals shown in (a) and (b) are representative of the two different outcomes obtained when the F1 cells were not displaced by the BALB/c lymphocytes. Data for the only animal in which the F1 cells were rejected, out of 15 in this group, are shown in (c). (d, e) In vivo proliferation: BALB/c nu/nu mice were injected on day 0 with F1 (d) or with BALB/c (e) splenocytes (30 × 106); 30 days later these mice received carboxi-fluorescein succinymidyl ester (CFSE)-labelled BALB/c adult thymocytes (20 × 106); in vivo proliferation of BALB/c thymocytes was analysed by monitoring CFSE dilution in spleen samples at day 7 after transfer; the proliferation cycles are indicated by boxes; the per cent distribution of cycling cells is indicated by numbers on each box.

The time interval between primary injection of F1 splenocytes and the subsequent transfer of BALB/c lymphocytes strongly influences the acquisition of tolerance in BALB/c nu/nu hosts

As described above, tolerance to the semi-allogeneic cells was obtained in BALB/c nu/nu hosts previously reconstituted with F1 splenocytes and further injected 30 days later with NSs, NTs or ATs. However, if these cells were injected alone into BALB/c nu/nu mice and allowed to reconstitute the host for 1 month, then in all cases these animals became immunocompetent and rejected a secondary injection of F1 splenocytes (Figs 1c,d and 4). To investigate the relevance of the time window for tolerance acquisition or effector response, we varied the time interval between the transfers of the two different populations. We then studied the outcome of a simultaneous injection of F1 and BALB/c splenocytes into BALB/c nu/nu hosts: the data obtained showed an intermediate result, with a clear reduction of the number of tolerant animals when compared with the protocol with the 30-day interval (F1 splenocytes on day 0, and then BALB/c on day 30) previously presented (Fig. 4).

Figure 4.

Figure 4

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The time interval between F1 peripheral colonization of BALB/c nude (nu/nu) mice and the transfer of BALB/c responder lymphocytes greatly influences the acquisition of tolerance. BALB/c nu/nu mice received 30 × 106F1 cells and 20 × 106 BALB/c adult thymocytes (AT), 30 × 106 neonatal thymocytes (NT) or 30 × 106 neonatal splenocytes (NS) on the same day (AT, n = 4; NT, n = 5; NS, n = 3), 30 days previously (NT, n = 4; NS, n = 7), or 30 days later (AT, n = 13; NT, n = 10; NS, n = 10). The percentage of tolerant animals was inferred from the persistence of F1 Thy-1.1+/Thy-1.2+ cells after flow cytometry analysis of blood samples at 30 days after the last transfer.

Tolerance acquisition in the lymphopenic (BALB/c × B6) F1 nude host

The result obtained above (Fig. 4) could indicate the need for a previous expansion of a subpopulation of F1 T cells (such as Tregs) for tolerance induction of the BALB/c T cells. Alternatively, the loss of fresh F1 APCs during the 30-day interval between transfers might not allow for an effective activation of alloreactive BALB/c T cells, leading to the preservation of the F1 cells. In order to investigate these possibilities we used (BALB/c × B6) F1 nu/nu mice as hosts, which allowed us to study tolerance induction of the different BALB/c populations in the absence of F1 T cells, and in an environment where F1 APCs are continuously replenished. Figure 5 shows that NTs, NSs and BALB/c ATs transferred to F1 nu/nu expanded equally and attained frequencies in the blood similar to those obtained in syngeneic nude hosts (Fig. 5a,b). However, only animals injected with neonatal populations (either thymocytes or splenocytes) were able to become tolerant to the host, as indicated by a low incidence of GVHD (Fig. 5b, insert), the lowest GVHD occurrence being obtained with thymocytes (21%). This tolerant state was confirmed by the prolonged maintenance of euthymic F1 splenocytes, transferred 1 month later to the same F1 nude mice previously injected with BALB/c NTs (not shown). Most BALB/c ATs, in contrast, were not tolerant to the F1 in this protocol and 75% of the group developed typical clinical signs of GVHD (reduction of body weight, protracted posture, dermatitis and chronic diarrhoea), culminating in death within 3–5 weeks.

Figure 5.

Figure 5

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Neonatal thymocytes/splenocytes and adult thymocytes are equally able to expand when transferred to F1 nude mice but only the neonatal populations acquire tolerance. F1 nu/nu hosts were injected with 20 × 106 adult thymocytes (AT→F1 nu/nu), 30 × 106 neonatal splenocytes (NS→F1 nu/nu), or 30 × 106 neonatal thymocytes (NT→F1 nu/nu). Expansion of BALB/c (Thy-1.2+) cells 1 month later was shown by flow cytometry of blood samples (a). The expansion of the same suspension of NTs in syngeneic BALB/c nu/nu hosts is shown as a control (NT→BALB/c nu/nu). Results obtained in individual mice of each group are shown in (b). Insert: numbers indicate the fraction and percentage of animals that died from GVHD/total F1 nudes that received BALB/c NTs (NT), neonatal splenocytes (NS) or adult thymocytes (AT).

Tolerance to semi-allogeneic stimuli in F1 (BALB/c × B6) nu/nu hosts does not affect the percentage of CD25+ Foxp3+ CD4+ T cells

We, and others, have previously shown that an elevated number of Tregs can inhibit alloreactivity.24–26 We thus investigated whether [BALB/c NT → (BALB/c × B6) F1 nu/nu] tolerant animals would have a higher frequency of Tregs compared with that obtained after expansion of the same cell population in syngeneic reconstituted (BALB/c NT → BALB/c nu/nu) mice. Figure 6(c–f) shows representative cytometric data for CD25/Foxp3 staining in peripheral blood of both groups of animals; for comparison, Fig. 6(a,b) shows the same CD25/Foxp3 staining for wild-type BALB/c mice. Figure 6(g,h) shows the compiled data for all the mice analysed. The frequencies of CD25+ Foxp3+ among CD4+ T cells in semi-allogeneic hosts (F1 nu/nu) were higher than those usually present in euthymic mice, but did not significantly differ from values observed in syngeneic BALB/c nu/nu hosts (Fig. 6g,h).

Figure 6.

Figure 6

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Neonatal thymocytes show similar frequencies of CD4+CD25+Foxp3+ cells after expansion in syngeneic and semi-allogeneic nude hosts. Flow cytometry analysis of CD4+CD25+ (a, c, e) and of CD25+Foxp3+ cells among CD4+ cells (b, d, f) in blood samples of euthymic BALB/c mice (a, b) or of BALB/c nude (nu/nu) (c, d) or F1 nu/nu (e, f) mice injected with 30 × 106 neonatal thymocytes 1 month previously is shown. (g, h) Percentages of CD4+ cells (g) and CD25+Foxp3+ cells among CD4+ cells (h) in blood samples of individual BALB/c nu/nu mice into which F1 adult splenocytes (AS) were transferred, BALB/c nu/nu mice into which BALB/c neonatal thymocytes (NT) were transferred, and F1 nu/nu mice into which BALB/c NT were transferred.

In the experimental model with [(BALB/c × B6.Ba) F1 → BALB/c nu/nu] mice, we also investigated whether Tregs would be enriched in the hosts 30 days after their transfer, before the injection of BALB/c T lymphocytes (Fig. 6g,h). The data obtained showed that the percentage of CD25+ Foxp3+ among CD4+ T cells was also higher than that observed in wild-type BALB/c animals (Fig. 6g,h) but comparable to that for the syngeneic reconstituted [BALB/c → BALB/c nu/nu] animals.

Discussion

In this study, we show that neonatal lymphocyte populations (thymus NT and spleen NS) and ATs can acquire tolerance to an allogeneic MHC haplotype, when transferred into a non-lymphopenic syngenic nude host, previously reconstituted with semi-allogeneic T cells. BALB/c NTs, NSs and ATs were for the most part unable to displace (BALB/c × B6.Ba) F1 T cells already established within the BALB/c nu/nu hosts; in a few animals both populations (BALB/c and F1) were observed 1 month after transfer. In both cases, specific tolerance to B6 skin grafts was established (Fig. 2).

The transferred BALB/c cells did not persist in most of the [(BALB/c × B6.Ba) F1 → BALB/c nu/nu] hosts, in spite of their initial proliferation, verified in the first week after transfer (Fig. 3). In most of these hosts, only the F1 cells could be detected after 1 month in blood, as well as in several peripheral organs, by immunofluorescence using anti-Thy-1.1/Thy-1.2 or anti-H-2Kd/H-2Db pairs of antibodies (Fig. 3a,b,c). These results indicate that the presence of F1 T cells does not impede the initial activation of the injected BALB/c cells but may prevent their further expansion and engraftment. It is interesting to note that BALB/c AT cells showed more intense proliferation in the chimeric host, probably because of the alloantigenic stimulation (Fig. 3e,g). NTs, NSs and ATs are naturally enriched for the presence of RTEs, and the existence of a time window of 3 to 4 weeks, during which RTE cells have no need to compete for surviving factors in the periphery, has been previously reported,27,28 which may explain the initial presence of transferred cells in the syngeneic BALB/c nude host and their proliferation. Their longer persistence depends on constant interactions with the MHC–peptide complex responsible for their survival,29–31 which makes the potential competition with F1 T cells for the same H-2d restricted MHC–peptide complexes an important obstacle to their homeostatic proliferation and engraftment, probably because the two populations share, to some extent, the same niche.

It is well established that homeostatic proliferation and engraftment are not observed when syngeneic mature T cells are transferred to non-lymphopenic hosts, a phenomenon also known as ‘isogenic barrier’. Recent studies on homeostatic proliferation revealed that mature T-cell clones can overcome this ‘barrier’ if their nominal antigen is present in the host.32,33 This seemed to be the case when BALB/c adult splenocytes were injected into [(BALB/c × B6.Ba) F1 → BALB/c nu/nu] mice, leading to complete elimination of F1 cells from the host, enabling the engraftment of adult BALB/c splenocytes. Even when very small inoculums of BALB/c splenocytes were used, containing numbers of CD4 and CD8 single-positive lymphocytes equivalent to those present in the RTE-enriched pools, F1 cells were excluded. In our protocol, the F1 resident cells of the nude host were maintained only in recipients of RTE-enriched populations. These results strongly suggest that the inability of NS, NT and AT cells to displace F1 cells was not attributable to an insufficient number of single positive CD4+ T cells, or to an absence of allogeneic stimuli, but was instead attributable to the functional state of these populations. Functional immunological immaturity of these populations has often been reported, especially concerning the poor generation of effector and memory T cells.34,35 It is thus possible that, for NT, NS and AT cells, the encounter of alloreactive clones with their nominal antigens would lead to initial proliferation (Fig. 3d) followed by their functional inactivation, anergy, and physiological elimination by apoptosis as a result of a weak response to antigenic stimuli. The apoptosis of anergic lymphocytes and their elimination by competition are well documented.36–40 We propose that, in the experiments described here, tolerance to allogeneic stimuli and difficulty with engraftment into non-lymphopenic hosts are associated: (i) BALB/c NT, NS and AT self-reactive T cells do not engraft in the F1-colonized peripheral compartment – the ‘isogenic barrier’– and BALB/c NT, NS and AT alloreactive T cells do not persist when tolerized; (ii) in contrast, adult BALB/c alloreactive T cells are functionally mature and the alloantigenic stimuli drive their activation, leading to the total elimination of F1 cells, which further allows the engraftment of self-reactive T cells.

It is important to note that NTs/lymphocytes and AT populations are not indefinitely incompetent to generate effective alloreactive T lymphocytes because, when previously transferred to the lymphopenic BALB/c nude host, they were fully able to reject the F1 splenocytes injected 1 month later (Fig. 1c,d). This protocol allows the colonization of the peripheral compartment with BALB/c NT, NS or AT cells before the injection of the semi-allogeneic F1 splenocytes, probably providing the necessary time period and environment for these populations to acquire immunological functional maturity. This hypothesis probably explains the correlation observed between the frequency of tolerant hosts and the time intervals between primary F1 colonization and the secondary transfer of BALB/c T cells (Fig. 4).

In addition to functional immaturity of the NT, NS and AT populations, other factors could be relevant in allowing tolerance induction in animals into which F1 splenocytes have previously been transferred. The 1-month interval between the two transfers might result in the depletion of fresh and professional F1 APCs or alter the nature and state of activation of these APCs, especially dendritic cells, which are crucial in determining the activation of alloreactive T lymphocytes (effector response versus tolerance).41 If tolerogenic or weakly immunogenic APCs predominate 1 month after the transfer of F1 cells, they may prevent the achievement of efficient alloreactive responses by BALB/c populations, especially for immature T cells. Interestingly, most lymphopenic F1 nudes injected with ATs died of GVHD, showing that neonatal populations have the unique property of acquiring tolerance in this system. The fact that ATs were mostly resistant to tolerance in F1 nu/nu, while they were tolerized in the [F1→ BALB/c nu/nu] chimeric animals, is indeed suggestive of a role for the abundance of professional APCs in tolerization. However, the tolerance of NTs and NSs obtained in F1 nude hosts, which have constant replenishment of semi-allogeneic APCs, suggests that such a mechanism does not interfere with tolerance of NTs or NSs. In these mice the expansion of BALB/c neonatal T-cell populations did not lead to GVHD in the majority of cases (Fig. 5b, insert). T cells are tolerized and persist indefinitely; the immunocompetence and specific tolerance of these animals were verified by the rejection of third-party skin grafts (not shown) and by the acceptance of splenocytes from euthymic F1 subsequently injected into these hosts (not shown).

It is important to note that these results obtained with the lymphopenic F1 nu/nu model provide empirical support for the possibility of T-cell engraftment in bone marrow transplantation or treatment of immunodeficient patients showing partial identity with donor MHC genes; manipulation of the immunological functional maturity of donor T cells could provide the key for successful reconstitution of immunocompetence without induction of GVHD. The data presented here extend previous studies on tolerance to minor histocompatibility antigens, using neonatal blood transplantation.42

Tregs, either naturally exported from the thymus or peripherally induced, are essential to control overt autoimmunity, and have also been implicated in neonatal acquired tolerance to alloantigens, including the Medawar system.2,3 The need for the previous establishment of the F1 cells in the periphery of the (BALB/c × B6.Ba) F1 →BALB/c nu/nu host, to tolerize BALB/c lymphocytes, could also indicate that the presence of a particular F1 T-cell population, such as Tregs, is required. As already demonstrated by Modigliani et al.,16 Tregs selected on an allogeneic thymic epithelium are able to convert RTE alloreactive cells to a tolerant and regulatory phenotype when both populations interact in the peripheral compartment. Here we investigated whether Tregs are enriched in [(BALB/c × B6.Ba) F1 → BALB/c nu/nu] mice, 30 days after their transfer, before the injection of BALB/c T lymphocytes. Although a higher percentage of CD25+ Foxp3+ cells was observed when compared with wild-type BALB/c animals, syngeneic reconstituted [BALB/c → BALB/c nu/nu] animals also had increased numbers of CD25+ Foxp3+ cells. We also investigated whether the transfer of neonatal populations to semi-allogeneic and lymphopenic F1 nude hosts, where donor BALB/c lymphocytes are tolerized and persist indefinitely, would result in an increased frequency of peripherally induced CD4+CD25+ Foxp3+ Tregs. The numbers of Tregs present 1 month after transfer were also higher than those observed in normal BALB/c and B6 mice. However, no significant differences were noticed when BALB/c NTs were transferred to syngeneic or to semi-allogeneic hosts, suggesting that the tolerance to B6 is not associated with a general increase in the total frequency of Tregs. These results contrast with our previous report on chimeric [(BALB/c × B6.Ba) F1 → BALB/c nu/nu] mice reconstituted with adult BALB/c splenocytes, where an enrichment for Tregs was found to be critical for tolerance. This discrepancy may be explained either by a more potent suppressive action of NT Tregs or by differences in the activation and functional maturation of naïve NT and naïve adult splenic T cells. Additionally, qualitative differences in the Treg repertoire in both hosts (syngeneic and semi-allogeneic) remain a possibility, such that the transfer of neonatal BALB/c T cells to the F1 nude host would result in enriched numbers of H-2b cross-reactive Tregs, without increasing their absolute numbers, which seem to be indexed to the total number of CD4+ CD25− naive T cells.43

The data presented here indicate an important contribution of peripheral mechanisms to the acquisition of tolerance by neonatal lymphocytes and AT populations. In contrast to adult mature lymphocytes, these cells could be rendered tolerant to allogeneic epitopes expressed by semi-allogeneic APCs present exclusively in the peripheral compartment, in the absence of the thymus, without the use of immunosuppression or enrichment for Tregs.

Acknowledgments

We thank Adriana Bonomo for stimulating discussions and encouragement. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa Carlos Chagas Filho (FAPERJ), Financiadora de Estudos e Projetos (FINEP) and Pro-Reitoria de Pesquisa e Pós-Graduação (PROPP-UFF), Brazil. The authors have no financial conflict of interest.

Abbreviations

AS

adult splenocytes

AT

adult thymocytes

APC

antigen-presenting cells

CFSE

carboxi-fluorescein succinymidyl ester

GVHD

graft-versus-host disease

MHC

major histocompatibility complex

NS

neonatal splenocytes

NT

neonatal thymocytes

PBS

phosphate-buffered saline

RTE

recent thymus-emigrant lymphocytes

Treg

regulatory T cell

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