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The Journal of Immunology, 1999, 162: 6926-6933.
Copyright © 1999 by The American Association of Immunologists

Cyclosporin A-Induced Autologous Graft-Versus-Host Disease: A Prototypical Model of Autoimmunity and Active (Dominant) Tolerance Coordinately Induced by Recent Thymic Emigrants1

Dianna Y. Wu and Irving Goldschneider2

Department of Pathology, School of Medicine, University of Connecticut Health Center, Farmington, CT 06030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cyclosporin A (CSA)-induced autologous graft-vs-host disease (autoGVHD) is an autoimmune syndrome initiated by autoeffector T cells presumed to be exported from the thymus during CSA treatment. The appearance of noncytotoxic immunoregulatory T cell activity after cessation of CSA treatment is also thymus dependent. In the present study, we have tested the hypothesis that both autoeffector and immunoregulatory T cells in CSA-treated rats are recent thymic emigrants (RTEs). Local syngeneic graft-vs-host reaction (synGVHR) and timed thymectomy (Tx) assays revealed that autoeffector T cells appear initially in the thymus and are promptly exported to lymph nodes (LN) during the first week of CSA treatment. In contrast, immunoregulatory thymocytes are first detectable by local synGVHR inhibition assays during the second week of CSA treatment but are not exported to LN until ~4 days post-CSA. Both the autoeffector and immunoregulatory T cells in LN express Thy-1, a selective marker for RTEs in the rat. However, the autoeffector RTEs have a CD4+8+ phenotype, whereas the immunoregulatory RTEs have a CD4+8- phenotype. Thus, the coordinate formation in and release from the thymus cortex and medulla of autoeffector and immunoregulatory T cells in CSA-treated rats directly demonstrates that centrally induced, nondeletional tolerance can serve as a fail-safe mechanism by which clones of autoeffector T cells that have escaped intrathymic negative selection for self-MHC class II Ag can be suppressed postthymically.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cyclosporin A (CSA)3 is an immunosuppressive agent widely used to treat autoimmune diseases, allograft rejection, and graft-vs-host disease (GVHD) (1). However, under certain experimental conditions, CSA can itself induce organ-specific autoimmunity and autologous GVHD (autoGVHD). For example, thyroiditis, gastritis, insulitis, oophoritis, and/or orchitis occur in certain strains of neonatal mice treated with CSA before thymectomy (Tx) and in athymic mice after engraftment of thymic lobes from CSA-treated syngeneic donors (2, 3). autoGVHD also occurs in adult rats treated with CSA either before Tx or after irradiation and syngeneic bone marrow transplantation (4, 5).

CSA-induced autoGVHD is mediated by autoeffector T cells that recognize self-MHC class II Ags (6). Generation of these autoeffector T cells has been linked to defects in both positive and negative selection of thymocytes, the former with interference in calcineurin activation (7) and the latter with alteration in the expression of MHC class II (and to a lesser extent MHC class I) molecules by thymic epithelial and dendritic cells (6). These and related defects result in the arrested development (8, 9) and massive release of cortical thymocytes (predominant phenotype in rats, TCR-{alpha}ßlow,CD4+CD8+,Thy-1high,TdT+) to peripheral lymphoid tissues during the first week of CSA treatment (10, 11, 12).

Inasmuch as thymocytes obtained during the first 12 days of CSA treatment, but not thereafter, can adoptively transfer GVHD to irradiated syngeneic recipients (13), it has been suggested that autoeffector T cells are exported to the peripheral lymphoid tissues soon after their generation in the thymus (14). Conversely, reconstitution of the thymic medulla after cessation of CSA treatment seems necessary for the prevention of autoGVHD (5), suggesting that the thymus may be involved in the formation and export of immunoregulatory T cells shortly after withdrawal of CSA. However, although the thymus is required for both the induction and prevention of autoGVHD, evidence of a role for recent thymic emigrants (RTEs) is indirect, especially given that low levels of autoeffector and immunoregulatory cells for MHC class II Ags can be found in the peripheral lymphoid tissues of normal rats (6).

To determine directly whether one or both of these T cell subsets is (are) formed in and exported from the thymus of CSA-treated rats, we used a quantitative local syngeneic graft-vs-host reaction (synGVHR) assay (15, 16) to trace the kinetics of the appearance of autoeffector and immunoregulatory cells in the thymus and lymph nodes (LN). We also used the Thy-1 Ag as a selective marker with which to identify RTEs among rat T cells (17). The results demonstrate that: 1) both autoeffector and immunoregulatory T cells in LN are RTEs; 2) autoeffector T cells are formed in and released from the thymus during the first week of CSA treatment; 3) immunoregulatory T cells are also formed in the thymus during CSA treatment but are not released to the periphery until cessation of CSA treatment; and 4) autoeffector RTEs have a CD4+8+ phenotype, whereas immunoregulatory RTEs have a CD4+8- phenotype. Thus, CSA-induced autoGVHD appears to be a prototypical model of both autoimmunity and nondeletional tolerance mediated by coordinately generated subsets of autoeffector and immunoregulatory RTEs from the thymus cortex and medulla, respectively.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Induction and diagnosis of autoGVHD

Female inbred Lewis (LEW) rats, 4–8 wk old, purchased from the National Cancer Institute (Frederick, MD), were used throughout this study. CSA in oral solution (generously provided by Sandoz, East Hanover, NJ) was mixed into standard rat chow in lieu of source at a concentration of 0.027% and pelleted (Dyets, Bethlehem, PA) (10). Rats were maintained ad libitum on either the CSA-containing food or the same food lacking CSA. The former protocol generates mean serum levels of CSA equivalent to those achieved by therapeutic doses administered parenterally (15 mg/kg body weight/day) (18).

To induce autoGVHD, rats were fed CSA for 14 to 18 days, thymectomized (Tx), and then fed normal food for an additional 14 to 15 days. Tx was performed under ether anesthesia, the sternum was divided in its superior portion, the prepericardial soft tissue including the thymus was removed by gentle suction, and the thorax and skin were closed with stainless steel wound clips (Becton Dickinson, Sparks, MD). Sham Tx rats were treated in a similar fashion, but the thymus was not removed. Acute GVHD was diagnosed at necropsy by histopathological examination of ear, skin, tongue, oral mucosa, main stem bronchi, and liver and were graded as to severity by standard criteria (19). More than 80% of positive animals exhibited grade 2 GVHD, as defined by intraepithelial lymphocyte infiltration in two or more organs (Table IGo).


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  		Table I. Effect of Tx on the development of autoGVHD in CSA-treated rats

 
Flow immunocytometric (FCM) analysis

Mouse mAbs to rat TCR-{alpha}ß (clone R73, FITC conjugated) and Thy-1.1 (clone OX-7, PE conjugated) were purchased from Harlan Bioproducts For Science (Indianapolis, IN). Single-cell suspensions of thymus (freed of adherent LN) and peripheral LN (pooled cervical, axillary, and inguinal) were made by gently pressing the lymphoid tissues through a 50 mesh stainless steel sieve into cold RPMI 1640 (Life Technologies, Grand Island, NY). One- or two-color labeling was performed by incubating 106 cells with FITC-conjugated and/or PE-conjugated Abs (17). Isotype-matched Abs were used as negative controls. All incubations were for 20 min at 4°C, after which the cells were washed with PBS containing 0.5% BSA and 0.1% sodium azide. Cells were processed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) equipped with an argon ion laser (488 nm), and data from immunofluorescence samples were analyzed using FACScan Research Software (Becton Dickinson). Lymphocytes were gated on the basis of forward and side scatter, and the percentage of T lymphocytes and Thy-1+ T cells among total lymphocytes was determined based on 104 events.

Immunomagnetic separation (IMS) of T lymphocyte subsets

To remove B cells, lymphocytes were treated at 4°C for 20 min with titrated mouse mAb to rat IgM (clone G53-238, biotin conjugated, PharMingen, San Diego, CA), washed twice, suspended in staining buffer (PBS with 5 mM EDTA and 0.01% sodium azide; 107 cells/90 µl), and treated with streptavidin-conjugated microbeads (Miltenyi Biotec, Sunnyvale, CA) at 6–12°C for 15 min (107 cells/10 µl). The cells were then washed twice with staining buffer, suspended in separation buffer (PBS with 5 mM EDTA, 0.5% BSA, 0.01% sodium azide; 107 cells/500 µl), and applied to the top of a separation column in a magnetic field (Vario MACS, Miltenyi Biotec). After several washes and backflushes with separation buffer, the T cell-enriched eluates (>95% purity as determined by FCM analysis for TCR-{alpha}ß) were pooled; stained for 20 min with mouse anti-rat Thy-1 (OX7), CD4 (W3/25), and/or CD8 (OX-8) mAbs (Harlan Bioproducts, Indianapolis, IN); washed; suspended in staining buffer (107 cells/80 µl); and incubated with goat anti-mouse IgG microbeads (Miltenyi Biotec) (107 cells/20 µl). Thy-1+ and Thy-1- T cells, as well as CD4 and/or CD8-enriched or depleted fractions, were then separated in a magnetic field, and the efficiency of separation (> 90%) was confirmed by FCM. The absence of Ab-mediated effects on T cell function was confirmed by comparing the activities of unseparated, Ab-treated LN cells with those of unseparated, untreated LN cells.

Quantitative local synGVHR assay

Induction. In preliminary experiments, the conditions of induction of a local hemiallogeneic GVHR in (LEW x BN)F1 rats (15) were optimized according to time of injection (7 days) and dose (linear between 0.5 x 106 and 16 x 106 cells) of LEW LN cells (data not shown). This assay was then adapted to detect the appearance of autoeffector T cells in CSA-treated rats, using a LEW -> LEW model of local synGVHR having similar optima. Thymocytes and/or LN cell suspensions from CSA-treated rats were injected s.c. (3 x 106 in 0.1 ml) into the right hind footpads of normal syngeneic recipients. Cell suspensions from untreated donors were injected into the left hind footpads of the same recipients to control for possible background autoeffector cell activity and nonspecific inflammation. Seven days after injection, the total number of T cells in each popliteal LN (PLN) was determined by FCM analysis, and the degree of local synGVHR was calculated according to the formula, local synGVHR index = no. of T cells in right (experimental) PLN/no. T cells in left (control) PLN.

Inhibition. In preliminary experiments, the conditions for inhibition of a local hemiallogeneic GVHR in (LEW x BN)F1 rats (20) were optimized according to time of injection (7 days) and dose of immunoregulatory cells (linear between 1.5 x 106 and 6 x 106 cells) from F1 rats immunized with LEW LN cells (data not shown). This assay was then adapted to detect immunoregulatory T cells from CSA-treated rats, as determined by inhibition of the local synGVHR. Thymocytes and/or LN cell suspensions from CSA-treated and control LEW rats were analyzed for immunoregulatory activity after being mixed with an equal number (3 x 106) of CSA day 5 LEW LN cells as a standardized source of autoeffector cells (see Results). The respective cell mixtures were injected in 0.1 ml into the right (experimental) and the left (control) footpads of normal syngeneic recipients, and the PLNs were harvested 7 days later. The percent inhibition of local synGVHR was calculated as % inhibition of local synGVHR = [(no. of T cells in left PLN - no. of T cells in right PLN) x 100]/no. of T cells in left PLN.

Blocking of local synGVHR

50 µl of 1:50 diluted mouse mAbs to rat RT1.B (clone OX-6; I-A determinant) and IgM (clone G53-238) as isotype control (PharMingen, San Diego, CA) were injected into right and left footpads of CSA day 7 LN recipients for 3 consecutive days, respectively, and the effects on the local synGVHR were observed as above.

Statistical analysis

All experiments were conducted with pooled cells from cohorts of three to four donors injected into groups of four recipients and were repeated two to four times. The significance of the differences in means between the number of T cells in right (experimental) and left (control) PLN or between Tx and sham Tx cohorts was determined by the paired t test. Except where noted, the SDs were <20% of the means.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Role of thymus in CSA-induced autoGVHR

To confirm that the thymus is necessary both for the induction and the prevention of autoGVHR in CSA-treated animals, rats were Tx or sham Tx before the onset, at the time of cessation, or 4 days after cessation of CSA treatment. The results in Table IGo show that only those rats that were Tx immediately at interruption of CSA treatment developed autoGVHR. As shown below, these results are best explained by the sequential export of autoeffector and immunoregulatory T cells from the thymus, the former occurring during CSA treatment and the latter occurring shortly after cessation of CSA treatment.

Kinetics of appearance of autoeffector cell activity in thymus and LN during and after CSA treatment

The kinetics of appearance of autoeffector cell activity in thymus and/or LN was determined in rats treated with CSA for up to 18 days. As shown in Fig. 1GoA, thymocytes from CSA day 1 through day 9, but not thereafter, were able to induce local GVHR in syngeneic recipients. Peak activity was reached on CSA day 4. In contrast, LN cells capable of inducing local synGVHR were first detected on CSA day 2, reached maximal levels by CSA day 7, and remained elevated through CSA day 18 (p > 0.1, CSA days 5 through 18).



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  		FIGURE 1. Kinetics of appearance of autoeffector cell activity in thymus and LN during and after cessation of CSA treatment. Experimental rats were fed CSA for up to 18 days (A) or CSA for 14 days and normal food for up to 15 additional days (B). Control rats were fed normal food only. Thymocytes or LN cells (3 x 106) obtained at the indicated times from experimental and control rats were injected into the right and left footpads of syngeneic recipients, respectively, and the relative levels of local synGVHR were determined 7 days later. Significant induction of local synGVHR (p < 0.05) in thymus (*) and LN (**) on days enclosed in brackets. Differences in vertical scales for A and B in this (and in Figs. 2Go and 3Go) represent experimental variation between different cohorts of rats analyzed during and after CSA treatment.

 
The fate of autoeffector cell activity after cessation of CSA treatment is shown in Fig. 1GoB. In these experiments, autoeffector activity failed to reappear in thymus and progressively decreased in LN between post-CSA days 0 and 15.

The specificity of the autoeffector T cells for self-MHC class II in the local synGVHR assay, as in the systemic synGVHD assay (21), was confirmed by mAb blocking studies (see Materials and Methods). As anticipated, injection of anti-RT1.B (I-A determinant) mAb into the footpads of recipients of CSA day 7 LN cells inhibited local synGVHR, whereas isotype control mAb did not (data not shown).

Role of thymus in appearance and persistence of autoeffector cell activity in LN during and after CSA treatment

To exclude the possibility that the appearance of autoeffector cells in LN during CSA treatment was unrelated to their initial appearance in thymus, rats were Tx before the onset of CSA treatment or at timed intervals thereafter. As anticipated, CSA day 12 and 18 LN cells from rats Tx on CSA day 0 failed to induce local synGVHR (data not shown; but see Table IGo, line 5). However, CSA day 12 and day 18 LN cells from rats Tx on CSA day 5 induced levels of local synGVHR equivalent to those of sham Tx controls (Fig. 2GoA), and Tx performed at the time of cessation of CSA treatment prevented the disappearance of autoeffector activity in post-CSA LN (Fig. 2GoB). Thus, the combined data in Figs. 1Go and 2Go suggest that the bulk of autoeffector cells in LN are exported from the thymus during the first week of CSA treatment but that the presence of the thymus after cessation of CSA inhibits the expression of autoeffector activity in LN, possibly by the exportation of immunoregulatory T cells.



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  		FIGURE 2. Effect of Tx during or on cessation of CSA treatment on the persistence of autoeffector cell activity in LN. Experimental rats were fed CSA for up to 18 days (A) or CSA for 14 days and normal food for up to 15 additional days (B). Control rats were fed normal food only. Rats were Tx or sham Tx on CSA day 5 or post-CSA day 0. LN cells (3 x 106) obtained at the indicated times from Tx or sham Tx experimental and control rats were injected into right and left footpads of syngeneic recipients, respectively, and the relative levels of local synGVHR were determined 7 days later. *, Significant difference between local synGVHR induced by LN cells from Tx and sham Tx rats (p < 0.05).

 
Kinetics of appearance of immunoregulatory cell activity in thymus and LN during and after CSA treatment

In these experiments, thymocytes and LN cells harvested from rats at timed intervals during and after CSA treatment were assayed for their ability to inhibit a local synGVHR induced by CSA day 5 LN cells (see Fig. 1GoA). The results in Fig. 3GoA show that thymocytes harvested between CSA days 7 and 14, but not day 3, exhibited significant inhibitory activity. This activity persisted in the thymus through post-CSA day 5, then abruptly disappeared (Fig. 3GoB). Conversely, no inhibitory activity was detected among LN cells at any time during CSA treatment (Fig. 3GoA), but such activity appeared abruptly in LN between post-CSA days 3 and 5, reached maximal levels by day 9, and persisted through at least day 15 (Fig. 3GoB).



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  		FIGURE 3. Kinetics of appearance of immunoregulatory cells in the thymus and LN during and after cessation of CSA treatment. Experimental rats were fed CSA for up to 14 days (A) or CSA for 14 days and normal food for up to 15 additional days (B). Control rats were fed normal food only. Thymocytes or LN cells (3 x 106) obtained at the indicated times from CSA-treated and control rats were mixed with 3 x 106 CSA day 5 LN cells as a source of autoeffector cells and injected into the right and left footpads of syngeneic recipients, respectively. The relative levels of inhibition of local synGVHR were determined 7 days later. *, Significant inhibition of local synGVHR (p < 0.05) in thymus (*) and LN (**) on days enclosed in brackets.

 
Prolongation of CSA treatment for 21 or 28 days proportionally extended the presence of immunoregulatory activity in the thymus, but cessation of CSA treatment still was required for the appearance of immunoregulatory cell activity in LN (data not shown).

Requirement of thymus for appearance of immunoregulatory cells in LN after CSA treatment

To exclude the possibility that the appearance of immunoregulatory cells in LN was unrelated to their initial appearance in thymus and to more precisely determine when immunoregulatory LN cells first appear after cessation of CSA treatment, rats were Tx on post-CSA days 0, 2, 4, or 6. LN cells were then harvested on post-CSA day 7 and assayed for their ability to inhibit the induction of local synGVHR. The results in Fig. 4Go show that as compared with LN cells from sham Tx rats, LN cells from rats Tx between post-CSA days 0 and 2, and to a lesser extent day 4, were deficient in immunoregulatory activity. However, LN cells from rats Tx or sham Tx on post-CSA day 6 had comparable immunoregulatory activities. Thus, by reference to Fig. 3GoB, it seems probable that immunoregulatory thymocytes begin to be exported to LN on or about post-CSA day 4.



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  		FIGURE 4. Effect of Tx on the appearance of immunoregulatory activity in LN after cessation of CSA treatment. Experimental rats were fed CSA for 14 days and normal food for 7 additional days. Control rats were fed normal food only. Rats were Tx or sham Tx at the indicated times, and, on post-CSA day 7, 3 x 106 LN cells from experimental and control rats were mixed with 3 x 106 CSA day 5 LN cells as a source of autoeffector cells and injected into the right and left footpads of syngeneic recipients, respectively. Relative levels of local synGVHR were determined 7 days later. *, Significant difference between inhibition of local synGVHR induced by LN cells from Tx and sham Tx rats (p < 0.05).

 
CSA-induced autoeffector and immunoregulatory LN cells are RTEs

Previous experiments have demonstrated that the Thy-1 Ag is a selective marker for RTEs among LN T cells in both normal and CSA-treated rats (10, 17). Hence, to confirm that CSA-induced autoeffector LN cells are RTEs (released from thymus <1 wk previously), >90% purified Thy-1+ and Thy-1- subsets of CSA day 4 LN T cells were isolated by IMS and tested by local synGVHR. As shown in Fig. 5Go, the autoeffector activity was exclusively recovered in the Thy-1+ T cell fraction. No autoeffector activity was detected in the Thy-1- T cell fraction or in the non-T cell fraction (data not shown).



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  		FIGURE 5. Autoeffector T cells that appear in LN during CSA treatment are Thy-1+. LN cells from rats fed CSA (experimental) or normal food (control) for 4 days were separated immunomagnetically into Thy-1+ and Thy-1- T cell fractions. Proportionate numbers of Thy-1+ T cells or Thy-1- T cells originally present among 4 x 106 LN cells were injected into the right (experimental) and left (control) footpads of syngeneic recipients. *, Significant induction of local synGVHR (p < 0.05).

 
Similarly, the immunoregulatory activity in post-CSA day 4 and post-CSA day 5 (data not shown) LN was isolated in the Thy-1+ T cell fraction (Fig. 6Go).



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  		FIGURE 6. Immunoregulatory T cells that appear in LN after cessation of CSA treatment are Thy-1+. Experimental rats were fed CSA for 14 days and normal food for 4 additional days. Control rats were fed normal food only. LN cells were separated into Thy-1+ and Thy-1- T cell fractions by IMS, and proportionate numbers of Thy-1+ T cells or Thy-1- T cells originally present among 4 x 106 LN cells were mixed with 3 x 106 CSA day 5 LN cells as a source of autoeffector cells and injected into the right (experimental) and left (control) footpads of syngeneic recipients. *, Significant inhibition of local synGVHR (p < 0.05).

 
CD4 and CD8 expression of autoeffector and immunoregulatory RTEs

The CD4/CD8 phenotype of autoeffector RTEs was determined by subjecting CSA day 4 LN T cells to IMS. As shown in Fig. 7Go, the bulk of the autoeffector activity was recovered in the CD4-enriched (CD4+8+ and CD4+8-), CD8-enriched (CD4+8+, CD4-8+), and CD4/CD8-enriched (CD4+8+, CD4+8-, and CD4-8+) fractions. However, autoeffector activity was markedly reduced in the CD4 and/or CD8-depleted fractions. Therefore, by exclusion, most of autoeffector T cells in CSA day 4 LN appeared to have a CD4+8+ (double-positive) phenotype.



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  		FIGURE 7. Phenotype of autoeffector T cells in CSA day 4 LN. LN cells from CSA day 4 and control rats were separated into CD4, CD8, or CD4/CD8-enriched or depleted T cell fractions by IMS. Proportionate numbers of each fraction originally present among 4 x 106 LN cells were injected into the right (experimental) and left (control) footpads of syngeneic recipients. *, Significant induction of local synGVHR (p < 0.05).

 
In contrast, the immunoregulatory activity was associated almost exclusively with CD4+ single-positive RTEs. Thus, as shown in Fig. 8Go, immunoregulatory T cell activity in post-CSA day 6 LN was significantly increased in the CD4-enriched, CD4/CD8-enriched, and CD8-depleted fractions and was significantly reduced in the CD4-depleted and CD4/CD8-depleted, as well as the CD8-enriched, fractions.



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  		FIGURE 8. Phenotype of immunoregulatory T cells in post-CSA day 6 LN. LN cells from post-CSA day 6 and control rats were separated into CD4, CD8, or CD4/CD8-enriched or depleted T cell fractions by IMS. Proportionate numbers of each fraction originally present among 4 x 106 LN cells were mixed with 3 x 106 CSA day 5 LN cells as a source of autoeffector cells and injected into the right (experimental) and left (control) footpads of syngeneic recipients. *, Significant inhibition of local synGVHR (p < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study directly documents the sequential export of autoeffector and immunoregulatory T cells from the thymus of CSA-treated and post-CSA-treated rats, respectively. The observation that autoeffector RTEs have a CD4+8+ phenotype when they first leave the thymus, whereas immunoregulatory RTEs have a CD4+8- phenotype, is consistent with our previous demonstration that short term CSA treatment (7 to 14 days) results in the export of large numbers of double-positive (CD4+8+) T cells from the thymic cortex, whereas cessation of CSA treatment results in the selective export of single-positive T cells from the regenerating thymus medulla (10, 11, 12). The results also suggest that these autoeffector and immunoregulatory cells are sequentially produced within the thymus during CSA treatment.

It is possible that the generation of immunoregulatory thymocytes is initiated somewhat earlier than CSA day 7 (Fig. 3Go) but that it is masked by the activity of autoeffector thymocytes (Fig. 1Go), just as the persistence or continued generation of autoeffector thymocytes after CSA day 9 may be masked by the activity of immunoregulatory thymocytes. However, our earlier demonstration that the export of RTEs from the thymus cortex (the likely origin of the autoeffector T cells) ceases entirely by CSA day 9 and does not resume substantially after cessation of CSA treatment (11, 12, 22) makes it unlikely that autoeffector thymocytes continue to be generated after the first week of CSA treatment. The reason for this remains to be determined, but the activities of the immunoregulatory thymocytes and the arrested maturation of newly generated cortical thymocytes during the second week of CSA treatment are obvious candidates (10). Conversely, it remains to be determined whether the presence of immunoregulatory thymocytes during CSA wk 3 and 4 is due to their continued generation and/or to their survival beyond CSA wk 2. What is clear is that immunoregulatory thymocytes are first exported to the periphery on or about post-CSA day 4. Furthermore, because Tx on post-CSA days 4 and 6 no longer promotes the development of autoGVHD, and because immunoregulatory activity no longer is detectable in the thymus after post-CSA day 5, it seems unlikely that the formation and/or release of immunoregulatory RTEs extends beyond the first week post-CSA.

It has been suggested elsewhere that the intrathymic generation of immunoregulatory cells in CSA-induced autoGVHD depends on the reconstruction of the medullary microenvironment after cessation of CSA treatment (6, 23). This seems improbable, given that the appearance of immunoregulatory thymocytes occurs during CSA treatment. However, because immunoregulatory thymocytes are not exported to the periphery until ~4 days after interruption of CSA treatment, we presume that they must undergo further intrathymic maturation before their release. This interpretation is consistent with reports that expression of extracellular matrix molecules such as fibronectin and recognition of MHC class II Ag, both of which are inhibited by CSA, may be necessary for final maturation and emigration of medullary thymocytes (24, 25).

Although the mechanism for the coordinate production of autoeffector and immunoregulatory thymocytes is not yet known, one possibility is that immunoregulatory thymocytes are formed in response to the appearance of autoeffector thymocytes. This hypothesis is supported by the observation that active tolerance to peripheral allografts can be induced by intrathymic inoculation of donor origin thymocytes or spleen cells in conjunction with brief immunosuppression (26, 27, 28, 29). It is also supported by evidence for TCR Id-anti-Id immunoregulation in allogeneic GVHD (30) and several models of organ-specific autoimmunity (31). As a variation, it is possible that both autoeffector and immunoregulatory thymocytes recognize self-MHC class II Ags but differ in their fine specificities for self peptide, the former being directed against self-MHC-peptide complexes on the autologous target cells, the latter against self-MHC-peptide complexes on the activated autoeffector T cells (32, 33, 34).

Alternatively, depending on the altered conditions of Ag presentation and thymocyte development (35, 36, 37), it is possible that subsets of MHC class II-specific immature thymocytes may differentially develop into autoeffector or immunoregulatory thymocytes. Thus, it has been observed that intrathymic recognition of MHC class II allopeptides is important for the induction of transplantation tolerance in rats and mice (38, 39) and that self-MHC class II expression, although significantly reduced during CSA treatment, still occurs on thymic epithelial cells and residual dendritic cells (40). It also is possible that, over time, autoeffector thymocytes may alter their cytokine profile to become immunoregulatory thymocytes. This has been suggested for peripheral T cells in anterior chamber-associated immune deviation (41).

Thus far, it has been shown that a public determinant of self-MHC class II Ags (I-A) is recognized by CD4-8+ autoeffector T cells in CSA-induced synGVHD and that a class II-associated invariant chain peptide is an enhancer for recognition of MHC class II+ target cells by Vß8.5+ autoeffector T cells (6). Furthermore, mAb blocking studies have suggested that the CD4+8- immunoregulatory T cells obtained from normal rats immunized with irradiated autoeffector T cells also recognize MHC class II molecules (6). Although it is not clear whether these immunoregulatory cells arise from preexisting T cells or are themselves RTEs induced by the immunization protocol, their Ag specificity and mode of action stand in contrast to those observed in allogeneic GVHD, in which CD4-8+ cytotoxic F1 T cells are thought to recognize the TCR-Id of alloeffector T cells (20, 30). This apparent disparity may reflect differences in the origins and times of formation of immunoregulatory T cells in the two systems, one population arising from preexisting peripheral T cells in response to the activation of parental alloeffector cells and the other arising in the thymus in concert with the spontaneous or induced formation of autoeffector T cells. It will be of especial interest, therefore, to determine the TCR Vß chain usage and mechanism of action of the immunoregulatory T cells in CSA-induced autoGVHD.

T cell tolerance to self Ags traditionally has been categorized as being either central (intrathymic) or peripheral (postthymic), depending on its origin and site of action. Central tolerance is primarily passive (recessive, deletional) (42, 43), whereas peripheral tolerance more often is active (dominant, nondeletional) (44, 45). Although this central/peripheral paradigm of T cell tolerance has been extremely useful, it has tended to obscure the existence of another major pathway of tolerance induction, namely centrally induced active peripheral tolerance. Thus, in other experiments involving the autoGVHD model (our manuscript in preparation), we have demonstrated that autoeffector T cells persist in LN for at least 4 wk after cessation of CSA treatment, even though they are not detectable functionally. Furthermore, these cells rapidly regain their functional activity when they are separated from immunoregulatory T cells, thereby indicating that the autoeffector T cells are not anergic and that the immunoregulatory RTEs are not cytotoxic.

Indirect evidence that immunoregulatory RTEs can induce active peripheral tolerance has long existed (46, 47, 48, 49). However, this notion has regained currency recently with the use of intrathymically injected Ag to manage graft rejection and autoimmunity (50, 51, 52, 53), and with the description of several experimental models of dominant tolerance (54, 55, 56). In addition, dysregulation in the production of autoeffector and immunoregulatory T cells has become a common theme in the pathogenesis of a variety of autoimmune disorders (22, 57, 58). Yet, to our knowledge, the present study is the first to directly demonstrate the coordinate development and export of autoeffector and immunoregulatory T cells in/from the thymus. If it is assumed that similar mechanisms explain the presence of autoeffector and immunoregulatory T cells for autoGVHD in the peripheral lymphoid tissues of normal adult mice (59), as well as the differential effects of timed Tx on the development and/or prevention of organ-specific autoimmunity in the neonatal period (60), then the present results suggest that immunoregulatory RTEs provide an important fail-safe mechanism whereby self-reactive T cell clones that have evaded intrathymic negative selection are prevented from initiating autoimmunity.

In addition to their role in down-regulating T cell responses to self-Ags, we have described two models of centrally induced peripheral tolerance in which RTEs down-regulate T cell responses to nonself Ags. Neither model requires thymic manipulation or dysfunction. In the first, injection of large doses of OVA in CFA in the hind footpad of rats causes the release of sequential waves of RTEs that inhibit the proliferation of effector T cells specific for PPD and OVA, respectively (Ref. 61; H. G. Durkin, S. M. Chice, and I.G., manuscript in preparation). In the second, injection of haptenated protein Ags into the anterior chamber of the eye of mice induces hapten-specific immune deviation (anterior chamber-associated immune deviation) by export of CD4-8- TCR-{alpha}ß+ immunoregulatory T cells from the thymus (62). These results suggest that by the induction of immunoregulatory RTEs on demand, dominant tolerance may play a key role in adaptive immunity to extrathymic nonself (and possibly self) Ags. Furthermore, the nature of the immunoregulatory RTEs may vary with the nature, dose, and route of injection of Ag. Consequently, as in autoimmunity, the full spectrum of involvement of immunoregulatory RTEs in adaptive immunity remains to be determined.


   Footnotes
 
1 This study was supported in part by National Institutes of Health Grant AI-33741. Back

2 Address correspondence and reprint requests to Dr. Irving Goldschneider, Department of Pathology, School of Medicine, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3105. E-mail address: Back

3 Abbreviations used in this paper: CSA, cyclosporin A; autoGVHD, autologous graft-vs-host disease; FCM, flow immunocytometry; IMS, immunomagnetic separation; LEW, Lewis; LN, lymph node; PLN, popliteal LN; RTEs, recent thymic emigrants; synGVHR, syngeneic graft-vs-host reaction; Tx, thymectomy. Back

Received for publication November 9, 1998. Accepted for publication March 17, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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