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Keywords:

  • Alopecia areata;
  • autoimmunity;
  • cyclosporine-A;
  • graft-versus-host disease;
  • regulatory T cells;
  • stem cell transplantation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

CD4 regulatory cells have been postulated to prevent autologous graft-vs.-host disease (GVHD). In order to test this hypothesis, we used BALB/c mice, a strain known to be resistant to autologous GVHD, which had received autologous stem cell transplantation (ASCT) and cyclosporine A (CsA). As expected, ASCT/CsA-treated BALB/c mice did not develop any sign of acute or chronic GVHD. However, depletion of CD4 T cells induced a skin disease with clinical and histological features of alopecia areata (AA), a CD8 T-cell-mediated human autoimmune skin disease. The hair loss in mice developing AA was associated with the infiltration of the skin by activated CD8 T cells. Analysis of the T-cell recovery in ASCT- and ASCT/CsA-treated mice showed that CsA induced an increase in the number of CD4+ 25+ T cells, suggesting that the lack of GVHD in ASCT/CsA treated-mice could be related to the expansion of this CD4 T-cell subset. Collectively these data show that CD4 T cells comprise regulatory cells controlling the onset of autologous GVHD and suggest that the naturally occurring CD4+ 25+ subset may be responsible for this effect.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Autologous stem cell transplantation (ASCT) is proposed as treatment of leukemia and lymphoma. Compared with allogeneic stem cell transplantation, its main advantage resides in the lower frequency of side-effects owing to the absence of graft-vs.-host disease (GVHD). However, lack of GVHD is associated with lack of graft vs. host leukemia (GVL) and with a higher relapse rate of malignancies in ASCT. In order to improve the antitumoral efficiency of ASCT, several groups have used different strategies to develop a controlled autologous GVHD, i.e. an immune reaction to self antigens with induction of autoantigen-specific B and T lymphocytes (1–4). Among those, cyclosporine (CsA) in association with ASCT has been widely used in animals and humans for inducing autologous GVHD with clinical features comparable to those observed during human alloGVHD (3,4). Models for CsA-induced autologous GVHD (autoGVHD) have been developed in rats and mice and have shown that: (i) autoGVHD develops in mice receiving a short course of CsA. Control animals receiving the diluent alone do not develop the disease (5); (ii) the clinical symptoms occur a few days after withdrawal of CsA (1,5); (iii) the clinical expression of autoGVHD varies greatly depending on the mouse strain and the protocol used. Acute autoGVHD may be fatal (6). Chronic autoGVHD may affect the gastro-intestinal tract or the skin (7); and (iv) development of the clinical symptoms of autoGVHD is owing to the activation in the tissues of autoreactive effector T cells which are under the control of CD4+ down-regulatory T cells (T reg) (8).

Although the mechanisms of action of CsA are not precisely known, several hypotheses have been proposed to explain how autoreactive T lymphocytes could develop into effector cells during ASCT. First, CsA is able to alter thymic epithelium and inhibit the expression of MHC molecules (9). It is thus possible that differentiating thymocytes expressing T-cell receptors directed to self-MHC molecules escape the negative selection and could thus be found as autoreactive T cells in the pool of peripheral naïve mature T cells. Second, CsA is able to interfere with the thymic differentiation of T-cell subsets and may alter the kinetics of recovery of effector vs. regulatory cells. In this respect, Wu and Goldschneider have shown that thymus emigration of CD4+ 25+ regulatory T cells in CsA-treated mice occurs several days after the emigration of mature naïve autoreactive T cells (8). This delay in CD4CD25 T-cell recovery may lead to a transient lack of down-regulatory mechanisms, allowing activation of effector T cells and development of autoGVHD.

In order to test the hypothesis that autoGVHD is an autoimmune disease owing to autoreactive effector T cells that are under the control of T reg, we have developed a model of autoGVHD in the BALB/c mouse strain which is not prone to develop CsA-induced autoGVHD. Indeed, several studies have shown that susceptibility to autoGVHD varied greatly from one strain to another. DBA/2 and C3H/He but not BALB/c mice are able to develop clinical symptoms following ASCT (6,10). BALB/c mice were chosen for this study because it is the most widely used mouse strain for the analysis of T-regulatory cells, which are considered to be particularly abundant and efficient in this mouse (11). Here, we show that BALB/c mice can be rendered sensitive to CsA-induced autoGVHD by depletion of CD4+ T cells, leading to the development of alopecia areata (AA), a CD8 T-cell-mediated autoimmune skin disease (12). These data confirm that CD4 regulatory cells can control the development of autoGVHD following ASCT.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Mice

Six-week-old female BALB/c and DBA/2 mice were used throughout this study. Female BALB/c and DBA/2 mice were purchased from Iffa Credo (L'arbresle, France).

ASCT-comprised conditioning regimen and injection of marrow stem cells (Figure 1)

image

Figure 1. Experimental protocol for induction of autologous graft-vs.-host disease.

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  • • 
    Conditioning regimen: mice received lethal total body irradiation (TBI) (850 rad).
  • • 
    Preparation and injection of bone marrow cells: BALB/c and DBA/2 mice were killed. Femurs and tibias were removed and bone marrow was flushed into sterile complete RPMI 1640 (Invitrogen Corporation, Paisley, UK) containing 5% FBS. Cells were collected, erythrocytes were removed by NH4Cl lysis and nucleated cells were washed twice. Between 0.8 and 1.107 nucleated Thy-1-depleted cells were injected into the tail vein to the syngeneic mice the day after irradiation.

Treatment by CsA after ASCT

Mice received 100-μL i.p. injections of CsA 20 mg/kg (Novartis, Rueil-Malmaison, France) (diluted in olive oil) or olive oil only, daily from day 1 to day 28 after ASCT (Figure 1).

Anti-CD4 and anti-CD8 mAbs treatment

Abs used in in vivo experiments comprised anti-CD4 and anti-CD8 mAbs, produced, respectively, by the hybridoma GK1.5 purchased from American Type Culture Collection (Manassas, VA) and the hybridoma H 35.17.2, kindly provided by G. Milon (Institut Pasteur, Paris, France). Some mice which were grafted received i.p. injections of 200 μL of anti-CD4 mAb and 200 μL of anti-CD8 mAb at various times after ASCT as 1/10 dilution of ascites fluid, twice per week from day 1 to day 60 after ASCT. Depletion of CD4+ and CD8+ T lymphocytes was checked by direct immunofluorescence and FACS analysis using anti-CD4 (clone CT-CD4) and anti-CD8 (clone CT-CD8a) mAbs (Caltag, Burlingame, CA, USA) (Figure 1).

Evaluation of autoGVHD

After withdrawal of CsA treatment, mice were observed daily and weighed three times weekly. Diarrhoea, hunched appearance, weight loss for three consecutive weighings or death consecutive to one of these features were considered as positive criteria of autoGVHD (6,10).

Cell preparation

Peripheral blood mononuclear cells were isolated by Ficoll gradient (Eurobio, Ulis, France). Single-cell suspensions of spleen and peripheral lymph nodes were made by gently pressing the lymphoid tissues through a 50-mesh stainless steel tissue into cold RPMI 1640.

FACS analysis

One, two or three-color labeling for CD4, CD8, CD3 (clone CT-CD3), CD25 (clone PC 61 5.3), CD62L (clone MEL-14) and CD69 (clone H1.2F3) (Caltag) was performed on fractions of lymph node cells, blood and spleen cells by incubating 106 cells with FITC, PE and Cy-Chrome-conjugating tricolor mAbs. Isotype-matched mAbs were used as negative controls. All incubations were for 20 min at 4°C, after which the cells were washed with buffer containing 0.5% BSA and 0.1% sodium azide. Cells were analyzed on FACScalibur flow cytometer (BD bio-sciences) equipped with an argon-laser (488 nm) and data from immunofluorescence samples were analyzed using CellQuest research software (BD bio-sciences). Lymphocytes were gated on forward and side-scatter and the percentage of cells belonging to each subset was determined on 10.103 events.

Histological analysis

Representative portions of normal and alopecic skin were removed and fixed in formaldehyde Bouin's fixative. Eight to 10-μm vertical sections were stained with hematoxylin-eosin and examined by light microscopy.

RT-PCR analysis

At different times after ASCT, skin samples were collected from mice and frozen into liquid nitrogen. Total RNA was extracted using a RNAXEL kit (Eurobio, Ulis, France), treated with DNase I, and 1 μg of total RNA was reverse-transcribed using poly (dT)15 primers and superscript II RT (Life Technologies, Cergy-Pontoise, France) for 90 min at 37°C. RNA detection was normalized using the housekeeping gene HPRT (hypoxanthine phosphoribosyltransferase) standard. The cDNA was then amplified using different sets of primers including: for HPRT (5′ primer, 5′-GTA ATG ATC AGT CAA CGG GGG AC-3′; 3′ primer, 5′-CCA GCA AGC TTG CAA CCT TAA CCA-3′), for CD8 (5′ primer, 5′-AGG ATG CTC TTG GCT CTT CC-3′; 3′ primer, 5′-TCA CAG GCG AAG TCC AAT CC-3′), and for IFN-γ (5′ primer, 5′-GCT CTG AGA CAA TGA ACG CT-3′, 3′ primer, 5′-AAA GAG ATA ATC TGG CTC TGC-3′). The amplifications were conducted: 29 cycles for HPRT, 33 cycles for CD8 and 35 cycles for IFN-γ (1 min at 94°C, 1 min at 61°C, and 1 min 30 at 72°C, respectively); and PCR products were analyzed on 1.5% agarose gel.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

BALB/c mice do not develop autoGVHD after ASCT and treatment by CsA

DBA/2 and BALB/c mice received the protocol of induction of autoGVHD as described in Figure 1 and were followed for 4 months. No sign of acute or chronic autoGVHD was seen in BALB/c mice before or after CsA withdrawal, confirming that this mouse strain is resistant to CsA-induced autoGVHD (Table 1). By contrast, clinical symptoms of autoGVHD, including diarrhoea, weight loss and hunched appearance, developed rapidly, within 7–15 days after withdrawal of CsA in DBA/2 mice. Death occurred after withdrawal of CsA in 5/11 DBA/2 mice. For mice that survived, the symptoms of autoGVHD progressively disappeared. This confirms that DBA/2 mice are sensitive to CsA-induced GVHD. Survivor mice of both strains were observed until day 120 after withdrawal of CsA without showing any other evocative features of autoGVHD.

Table 1.  Clinical features of autologous graft-vs.-host disease in mice after autologous stem cell transplantation
  Diarrhoea Weight lossHunched appearanceDeath after CsA withdrawalTotal CsA-induced autoGVHD
  1. For a total of eight BALB/c mice transplanted none developed evocative clinical features of autoGVHD, whereas seven out 11 DBA/2 mice developed autograft-vs.-host disease.

BALB/c strain0/80/80/80/80/8
DBA/2 strain2/117/115/115/117/11

CD4 T-cell depletion after ASCT induces a chronic autoGVHD in BALB/c mice

As CD4 T-regulatory cells have been postulated to prevent autoGVHD (8,13), we next tested the effect of CD4 T-cell depletion, and as control the effect of CD8 T-cell depletion, in the development of autoGVHD in BALB/c mice receiving ASCT/CsA. CD4 (or CD8) T-cell depletion was maintained over 2 months (day 1 to day 60) by twice/week infusion of the depleting anti-CD4 (or CD8) mAb. The magnitude of CD4 or CD8 T-cell depletion was checked once a week by FACS analysis and residual T-cell blood levels were found to be less than 5% of the normal value throughout the treatment period.

No changes were observed in mice treated by ASCT/CsA and anti-CD8 mAbs during the 60 days of treatment and the 2 months of follow up.

Alternatively, CD4 T-cell-depleted, ASCT/CsA-treated BALB/c mice developed AA, an autoimmune skin disease, owing to the destruction of hair follicles by skin infiltrating CD8 T lymphocytes (12,14). The skin changes typical of experimental AA started to appear at day 45 in mice treated by anti-CD4 mAb, were maximal at day 60 (i.e. the end of anti-CD4 mAb injections) and were absent in the control ASCT/CsA-treated mice (Figures 2A,B). Alopecia areata lesions developed slowly from small bald patches on the head (day 45) to large loss of hair on the dorsal skin (day 60). The bald skin had a normal appearance without any sign of inflammation. The extent of the alopecia was not affected by the withdrawal of anti-CD4 mAb treatment and persisted without major changes during the 2-month follow up after the end of the treatment. Histological examination of alopecic skin of the ASCT/CsA/anti-CD4 mAb-treated (Figure 2D) compared with the normal ASCT/CsA-treated BALB/c (Figure 2C) mice revealed changes compatible with AA (15), showing: (i) a reduced number of hair follicles; (ii) a smaller size of hair follicles; and (iii) a diffuse and moderate lymphocytic infiltration found around hair follicles and extending to the lower surface epidermis (Figure 2D). Alternatively, the sebaceous glands, which are appended to the hair follicles, were not altered in their size or appearance, demonstrating that hair follicles only are affected in ASCT/CsA/anti-CD4 mAb-treated mice.

image

Figure 2. Development of alopecia areata in CD4+ T-cell-depleted, autologous stem cell transplantation (ASCT)/CsA-treated BALB/c mice. Alopecia areata skin lesions occurred as soon as day 45 (A) and were more diffuse at day 60 (B). Histological analysis was performed on normal-looking skin of ASCT/CsA-treated BALB/c mice (C) and on alopecic skin of ASCT/CsA/anti-CD4 mAb-treated BALB/c mice (D). Alopecic skin showed a diffuse and moderate dermal lymphocytic infiltration [[RIGHTWARDS ARROW]], which predominated around hair follicles and extending to the lower surface epidermis, reduced size and number of follicles [▴], whereas the sebaceous glands which are appended to the hair follicles were not altered [[RIGHTWARDS DOUBLE ARROW]]. E, epidermis; D, dermis.

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We observed no sign of clinical abnormalities that would suggest the existence of other pathologies in both ASCT/CsA- and ASCT/CsA/anti-CD4 mAb-treated mice.

These data show that CD4 T-cell depletion render BALB/c mice susceptible to autoGVHD, which expresses as an autoimmune skin disease, i.e. Alopecia areata (Table 2). Because the skin lesions start approximately 45 days after ASCT we defined this autoGVHD as chronic. It is noteworthy that CsA was necessary for the expression of chronic autoGVHD in BALB/c mice receiving anti-CD4 mAb treatment, as no sign of AA was observed in the group of mice receiving ASCT and anti-CD4 mAb treatment only.

Table 2.  Requirements for generation of alopecia areata in BALB/c mice
 Total no. of miceAlopecia areataWeight lossHunched appearanceDeath after mAb withdrawal
  1. Respective numbers of mice that developed clinical features after particular experimental immune manipulation are shown. It appears that only mice treated by ASCT/CsA and anti-CD4 mAbs developed inflammatory skin disease (alopecia areata).

ASCT100100
ASCT + CsA100100
ASCT + CsA + anti-CD4 mAb108200
ASCT + CsA + anti-CD8 mAb50000
ASCT + anti-CD4 mAb50000
CsA + anti-CD4 mAb50100

Chronic autoGVHD is associated with skin infiltration by CD8 T cells

As human and experimental AA is associated with the infiltration of hair follicles by activated CD8+ T cells (16), we next examined the presence of CD8 T cells in the skin of BALB/c mice receiving ASCT/CsA with and without anti-CD4 mAb treatment, using semi quantitative RT-PCR analysis for CD8 and IFNγ mRNAs (Figure 3). We have previously shown that recruitment of activated CD8+ T cells in murine skin during development of antigen-specific inflammation could be followed by RT-PCR analysis for CD8 and IFNγ mRNA (17). CD8 and IFNγ mRNA was not found in the skin of normal mice, as previously described (18) or in the skin of mice receiving ASCT/CsA only at any time point studied. Conversely, prominent infiltration of CD8 T cells was found in the alopecic skin of mice receiving ASCT/CsA+ anti-CD4 mAb treatment at day 60 (corresponding to the peak of AA lesions), whereas no such infiltration was noted at day 35 (i.e. a week before the onset of AA lesions) (Figure 3). Interestingly, CD8 T-cell infiltration was also found in the normal looking skin of alopecic mice, although to a lesser extent, suggesting the occurrence of a diffuse immune response in the skin of those mice.

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Figure 3. RT-PCR analysis of CD8 and γIFN mRNA in skin of BALBc mice. CD8 and IFN-γ mRNA expression was analyzed using semiquantitative RT-PCR analysis of biopsies of dorsal skin obtained from normal (lane 1), autologous stem-cell transplantation (ASCT)-treated (lanes 2 and 3) and ASCT/CsA-treated (lanes 4 and 5) BALB/c mice, as well as in nonalopecic (lane 6, day 36 and lane 7, day 60) and alopecic skin (lane 8, day 60) of ASCT/CsA/anti-CD4 mAb-treated BALB/c mice.

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These data show that development of AA in BALB/c mice correlates with skin infiltration by activated CD8 T cells; a feature typical of the human AA disease.

Immune reconstitution of ASCT- treated BALB/c mice

The conditioning regimen used in this study comprised a short course of CsA which has been shown previously to induce autoGVHD in DBA/2 mice (1). This was not the case in BALB/c mice for which CD4 T-cell depletion was necessary to induce autoGVHD. In order to understand why BALB/c mice did not develop autoGVHD after ASCT/CsA treatment only, we next compared the T-cell reconstitution in BALB/c and DBA/2 mice.

In both strains, ASCT induced a profound T-cell lymphopenia which was observed from day 1 to day 15. From day 15 to day 35, the number of CD3, CD4 and CD8 T cells increased regularly up to normal levels and the percentage of CD4+ 25+ T cells was approximately 20% of the number of CD4 T cells. After ASCT/CsA, the reconstitution of CD8 T cells was similar to that obtained with ASCT only, but major differences occurred in CD4 T-cell recovery. First, reconstitution of CD4 T cells was delayed and obtained at day 50 only. From day 28 to day 42, the absolute number of CD4 T-cell was half that observed in ASCT-treated mice (data not shown). Secondly, the percentage of CD4+ 25+ T cells among total CD4+ T cells was higher in ASCT/CsA-treated BALB/c compared with DBA/2 mice from day 28 to day 42. CD4+ 25+ T cells represented up to half of the number of CD4 T cells in BALB/c mice at day 28, whereas in DBA/2 mice they represented less than 25% (Figure 4). These CD4+ 25+ T cells did not express the early activation marker CD69 and were CD62L positive, suggesting that they do not represent activated T cells but more likely correspond to the naturally occurring T-regulatory cells. These data show that CsA dramatically alters the quantitative and qualitative reconstitution of CD4 T cells in ASCT-treated mice, resulting in the overrepresentation of CD4+ 25+ T cells among total CD4 T cells in BALB/c and a to a lesser extend in DBA/2 mice. It is thus tempting to speculate that lack of autoGVHD in BALB/c mice following ASCT/CsA is owing to the higher proportion of CD4CD25 T cells found in this mouse.

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Figure 4. Recovery of CD4+ 25+ T lymphocytes after autologous stem cell transplantation (ASCT) and ASCT/CsA in BALB/c mice. Quantitative recovery of CD4+ 25+ lymphocytes was performed in both DBA/2 and BALB/c mice by FACS analysis of total lymph node cells (gate on the lymphocyte population) double-stained for CD4 and CD25 using specific mAbs, every week from day 14 until day 42 after ASCT. The data presented here are representative of three experiments. Note the difference in the percentage of CD4+ CD25+ T cells between BALB/c mice and DBA/2 mice at day 28 (circles).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In this study, we have shown that CD4 T cells prevent the development of chronic autoGVHD in BALB/c mice. Indeed, ASCT/CsA-treated BALB/c mice that are normally resistant to autoGVHD can develop AA, an autoimmune skin disease, when CD4 T cells are removed. Alopecia areata is an autoimmune skin disease resulting in hair loss which is secondary to the infiltration of hair follicles by T cells (15,16). Although both CD4 and CD8 T cells are prominent in the infiltrates, studies in the Dundee bald rat model for AA have shown that CD8 T cells were effector cells, as in vivo depletion of CD8 T cells restores hair growth in these rats (12). More recently, Gilhar et al. extended these observations to the human disease by showing that AA was transferable to human scalp grafts on SCID mice by injection of autologous-activated lesional T cells recovered from the patient's AA lesions (16). Pathogenic γIFN-producing T cells migrated into the grafted scalp and induced hair loss by a mechanism which is thought to associate T-cell cytotoxic activity towards follicular epithelial cells and production of inflammatory cytokines (18). In our study, the clinical and histological features of experimental AA obtained in CD4-depleted ASCT/CsA-treated BALB/c mice were similar to those of the human disease and of other animal models of AA (16,19,20). Hair loss appeared to be secondary to the infiltration of hair follicles by γIFN-producing CD8 T cells. It is noteworthy that BALB/c mice are not prone to develop AA either spontaneously (19) nor after in vivo CD4 T-cell depletion. In this study, occurrence of AA was observed only in mice that received ASCT and CsA, suggesting that, during the immune recovery, follicular autoantigen-specific CD8 T cells are primed but cannot exert their effector function because they are under the control of regulatory/suppressor CD4+ T cells. Thus development of AA during ASCT/CsA and anti-CD4 mAb corresponds to a manifestation of chronic autoGVHD in BALB/c mice.

Graft-vs.-host disease normally develops in clinical settings when allogeneic tissues containing immunocompetent cells are transferred to an immunocompromized host; a frequent scenario in allogeneic SCT (21). However, GVHD can also develop after autologous or syngeneic stem cell transplantation, even though donor and recipient express identical tissue antigens (1–4). Human GVHD has occurred in recipients of syngeneic bone marrow from an identical twin (22). However, the frequency of GVHD after ASCT in humans remains low. Administration of CsA following ASCT results in an increase in the frequency and the intensity of GVHD (3,4) and most of the studies on experimental autoGVHD use CsA (23). The protocol applied to generate autoGVHD in the present study has been widely used and consisted of total body irradiation, ASCT and a 4-week course of CsA. In mice, CsA is necessary for the development of autoGVHD, as animals receiving ASCT alone almost never develop pathological clinical symptoms. Recent studies have brought new insights into the mechanisms of action of CsA following ASCT by demonstrating that CsA is able to delay the emigration of CD4+ 25+ T-regulatory cells without affecting the departure of other T-cell subsets (8). It is thus tempting to speculate that, when CsA treatment is stopped, mature CD8+ and CD4+ 25– T cells can be activated and become effector cells leading to autoGVHD, the more so because T-regulatory cells still reside in the thymus. Following withdrawal of CsA, the CD4 T-reg pool emigrates out of the thymus and in a few days reconstitutes the host and can suppress the effector T cells (8,13). This may explain why the clinical symptoms of autoGVHD are transient and followed by a complete recovery, provided that the recipients do not die of a severe acute GVHD. This is what was observed in our study of autoGVHD in the susceptible DBA/2 mice where seven mice out of 11 developed acute autoGVHD, which was fatal in five of them. The two mice that recovered from the disease displayed a normal phenotype and remained in good health.

However, CsA treatment associated with ASCT is not always sufficient to induce autoGVHD in all strains of mice. In the present study, BALB/c mice tolerated the ASCT+ CsA protocol and did not develop any acute or chronic symptoms reminiscent of autoimmunity. Although it is known that some strains of mice are resistant whereas others are susceptible to CsA, the reasons for these differences remain unknown. As the occurrence of autoGVHD in susceptible mice has been linked to a transient defect in the recovery of T-regulatory cells, it is thus likely that the lack of autoGVHD in resistant mice is owing to the presence of peripheral T-regulatory cells at the time of the emigration of autoreactive T cells out of the thymus. Along this line, recent studies by Barendrecht et al. have shown that susceptibility compared with resistance to CsA-induced autoimmunity in different rat strains depends on the number of peripheral CD4 T-regulatory cells expressing CD45RClow (24). Our data analyzing the CD4 T-cell pool and the relative proportion of CD4CD25+ T cells among total CD4+ T cells are in keeping with this hypothesis. While CsA treatment dramatically decreased the number of CD4 T cells found in the blood and secondary lymphoid organs of ASCT-treated BALB/c and DBA/2 mice, the percentage of CD4+ 25+ T cells among total CD4 T cells was markedly enhanced in BALB/c compared with DBA/2 mice. Thus, at day 28, i.e. during CsA withdrawal, CD4+ 25+ T cells comprised 50% of total CD4 T cells in ASCT/CsA-treated BALB/c mice compared with 21% in ASCT/CsA-treated DBA/2 mice. Furthermore, at day 42 CD4+ 25+ T cells comprised 17% of total CD4 T cells in ASCT/CsA-treated BALB/c mice compared with 6% in ASCT/CsA-treated DBA/2 mice. It is thus possible that the increase in the CD4CD25 T-regulatory-cell population among the CD4 T-cell pool is responsible for an efficient suppression of CD8 effector cells in BALB/c mice.

That CD4 T cells endowed with down-regulatory activity behave as suppressor cells of autoGVHD is demonstrated by our experiments of in vivo depletion of CD4 T cells in ASCT/CsA-treated BALB/c mice which thus develop skin autoimmunity. It is noteworthy that CsA is necessary for the occurrence of chronic GVHD, as ASCT-treated, CD4 T-cell-depleted animals do not show any sign of autoGVHD. These results suggest that the effect of CsA on the induction of autoGVHD cannot solely be explained by a retention in the thymus of T-regulatory cells while effector T cells are released at the periphery. It is likely that CsA also acts on the development of effector T cells and that CsA treatment leads to the differentiation of autoreactive effector T cells which would have been deleted in the absence of CsA (2,25,26). In this respect, Teshima et al. have recently shown that impaired thymic negative selection, which occurs in MHC class II-deficient C57BL/6 mice, can cause autoimmune GVHD which, in this mouse strain, is mediated by autoreactive CD4 T cells and down-regulated by CD4 and CD8 T-regulatory cells (27). The authors postulate that the lack of MHC class II molecules leads to the survival and export of the periphery of autoreactive T cells which can thus be activated and induce the disease. As CsA treatment markedly reduces expression of MHC molecules on thymic stromal cells (9) it is likely that negative selection of autoreactive T cells is hampered in ASCT/CsA-treated mice. It is thus possible that in ASCT-treated BALB/c mice, CsA is responsible for impaired negative selection of autoreactive CD8 T cells which are exported at the periphery but cannot induce immunopathology because CD4 T-regulatory cells are present and suppress their activation.

In conclusion our data highlight the pivotal role of CD4 regulatory T cells in the maintenance of tolerance to self antigens. Several subsets of regulatory CD4 T cells have been characterized in humans and mice (13,28) and their relative contribution to transplantation tolerance is beginning to be studied (29). However, manipulation of the T-regulatory cells may open new avenues in the treatment of patients receiving stem-cell transplantation for malignancies.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work was supported by La Ligue Contre le Cancer du Rhône and La Région Rhone-Alpes (agreement # 8HC07H). We are indebted to Ms Rosine Tedone, Josette Benetière, Marie Thérèse Ducluzeau and Magalie Valeyrie for excellent technical assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Glazier A, Tutschka PJ, Farmer ER, Santos GW. Graft-versus-host disease in cyclosporin A-treated rats after syngeneic and autologous bone marrow transplantation reconstitution. J Exp Med 1983; 158: 18.
  • 2
    Hess AD, Horwitz LR, Beschorner WE, Santos GW. Development of graft-vs-host disease-like syndrome in cyclosporine-treated rats after syngeneic bone marrow transplantation. Development of cytotoxic T-lymphocytes with apparent polyclonal anti-Ia specificity, including autoreactivity. J Exp Med 1985; 161: 718730.
  • 3
    Jones RJ, Hess AD, Mann RB et al. Induction of graft-versus-host disease after autologous bone marrow transplantation. Lancet 1989; 1: 754757.
  • 4
    Kennedy MJ, Vogelsand GB, Jones RJ et al. Phase I trial of interferon gamma to potentiate cyclosporine-induced graft versus-host disease in women undergoing autologous bone marrow transplantation. J Clin Oncol 1994; 12: 249257.
  • 5
    Sorokin R, Kimura H, Shroder K, Wilson DH, Wilson DB. Cyclosporine-induced autoimmunity. Conditions for expressing disease, requirement for intact thymus and potency estimates of autoimmune lymphocytes in drug-treated rats. J Exp Med 1986; 164: 16151625.
  • 6
    Bryson JS, Jennings CD, Caywood BE, Kaplan AM. Induction of a syngeneic graft-versus-host disease-like syndrome in DBA/2 mice. Transplantation 1989; 48: 10421047.
  • 7
    Osman Y, Watanabe T, Kawachi Y et al. Intermediate TCR cells with self-reactive clones are effector cells which induce graft-versus-host disease in mice. Cell Immunol 1995; 166: 172186.
  • 8
    Wu DY, Goldschneider I. Tolerance to cyclosporin A-induced autologous graft-versus-host disease is mediated by a CD4+CD25+ subset of recent thymic emigrants. J Immunol 2001; 166: 71587164.
  • 9
    Shinozawa T, Beschorner WE, Hess AD. The thymus and prolonged administration of cyclosporine: irreversible immunopathologic changes associated with autologous pseudo-graft-vs-host-disease. Transplantation 1990; 50: 106111.
  • 10
    Bryson JS, Jennings CD, Caywood RI, Kaplan AM. Strain specificity in the induction of syngeneic graft-versus-host disease in mice. Transplantation 1991; 51: 911913.
  • 11
    Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor α-chains (CD25)Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995; 155: 11511164.
  • 12
    McElwee KJ, Spiers EM, Oliver RF. In vivo depletion of CD8+ T cells restores hair growth in the DEBR model for alopecia areata. Br J Dermatol 1996; 135: 211217.
  • 13
    Hess AD, Fischer AC, Horwitz L, Bright EC, Laulis MK. Characterization of peripheral autoregulatory mechanisms that prevent development of cyclosporin-induced syngeneic graft-versus-host disease. J Immunol 1994; 153: 400411.
  • 14
    Hoffmann R. The potential role of cytokines and T cells in alopecia areata. J Invest Dermatol Symp Proc 1999; 4: 235238.
  • 15
    Perret C, Weisner-Menzel L, Happle R. Immunohistochemical analysis of the T-cell subsets in the peribulbar and intrabulbar infiltrates of alopecia areata. Acta Derm Venereol 1984; 64: 2640.
  • 16
    Gilhar A, Shalaginov R, Assy B, Serafimovich S, Kalish RS. Alopecia areata is a T-lymphocyte mediated autoimmune disease: lesional human T-lymphocytes transfer alopecia areata to human skin grafts on SCID mice. J Invest Dermatol Symp Proc 1999; 4: 207210.
  • 17
    Akiba H, Kehren J, Ducluzeau MT et al. Skin inflammation during contact hypersensitivity is mediated by early recruitment of CD8+ T cytotoxic 1 cells inducing keratinocyte apoptosis. J Immunol 2002; 168: 30793087.
  • 18
    Gilhar A, Landau M, Assy B et al. Transfer of alopecia areata in the human scalp graft/Prkdcscid (SCID) mouse system is characterized by a TH1 response. Clin Immunol 2003; 106: 181187.
  • 19
    McElwee K, Freyschmidt-Paul P, Ziegler A, Happle R, Hoffmann R. Genetic susceptibility and severity of alopecia areata in human and animal models. Eur J Dermatol 2001; 11: 1116.
  • 20
    Freyschmidt-Paul P, Ziegler A, McElwee KJ et al. Treatment of alopecia areata in C3H/HeJ mice with the topical immunosuppressant FK506 (Tacrolimus). Eur J Dermatol 2001; 11: 405409.
  • 21
    Ferrara LM, Teshima T. Understanding the alloresponse: new approach to graft-versus-host disease prevention. Semin Hematol 2002; 39: 1522.
  • 22
    Rappeport J, Mihm M, Reinherz E, Lopanski S, Parkman R. Acute graft-versus-host disease in recipients of bone-marrow transplants from identical twin donors. Lancet 1979; 2: 717720.
  • 23
    Hess AD, Thoburn CJ. Immunobiology and immunotherapeutic implications of syngeneic/autologous graft-versus-host disease. Immunol Rev 1997; 157: 111123.
  • 24
    Barendrecht MM, Tervaert JW, Van Breda Vriesman PJ, Damoiseaux JG. Susceptibility to cyclosporin A-induced autoimmunity: strain differences in relation to autoregulatory T cells. J Autoimmune 2002; 18: 3948.
  • 25
    Urdahl KB, Pardoll DM, Jenkins MK. Cyclosporin A inhibits positive selection and delays negative selection in αβ TCR transgenic mice. J Immunol 1994; 152: 28532859.
  • 26
    Severino ME, Laulis MK, Horwitz LR, Hess AD. Cyclosporine preferentially inhibits clonal deletion of CD8 positive T-cells with an MHC class II restricted autoreactive T-cell receptor. Transplant Proc 1993; 25: 520523.
  • 27
    Teshima T, Reddy P, Liu C, Williams D, Cooke KR, Ferrara LM. Impaired thymic negative selection causes autoimmune graft-versus-host disease. Blood 2003; 102: 429435.
  • 28
    Dubois B, Chapat L, Goubier A, Kaiserlian D. CD4+CD25+ T cells as key regulators of immune responses. Eur J Dermatol 2003; 13: 111116.
  • 29
    Jiang S, Lechler RI. Regulatory T cells in the control of transplantation tolerance and autoimmunity. Am J Transplant 2003; 3: 516524.