T cell repertoire expression in murine recipients of bone marrow transplant after LF 08-0299 (Tresperimus) administration

Transplant Immunology 1998; 6: 217-224

T cell repertoire expression in murine recipients of bone marrow transplant after LF 08-0299 (Tresperimus) administration Jocelyne Annat and Patrick Dutartre Immunology Department, Laboratoires Foumieq Daix Received 23 September 1998; revised version accepted 24 November 1998

Ah&rack LF 08-0299 (Tkesperimus), a novel immunosuppressive compound, has been previously shown to prevent graft-versus-host disease in murine models. In this study, we investigated the tiuence of LF 080299 on the TCR VP repertoire of irradiated Fl recipient mice reconstituted with either syngeneic or parental bone marrow cells. We showed that a partial blockade of thymic differentiation occurred in normal mice under treatment at the transition C.D4-/CD8- to CD4+/CD8+, and that this blockade was fully reversible. Despite the effect on the thymus, normal T cell repertoire negative selection was preserved following syngeneic bone marrow transplantation. We further assessed whether LF 08-0299 administration could modify VP T cell expression in irradiated recipients reconstituted with parental bone marrow cells. In our murine parental to Fl transplant model, abnormal TCR Vp3, Vfi5, VP6 and VP11 expression was demonstrated in peripheral lymph nodes of irradiated recipients. Moreover, Vfl6 and VP3 T cell populations were overexpressed. Administration of LF 08-0299 modified the pattern of Vfl T cell expression. The expansion of V/36 T cells was selectively inhibited under LF 08-0299 therapy and, in contrast, VP5 T cells were overexpressed. Lymph node histological analysis showed that LF 08-0299 administration fully prevented fhe graft-versus-host reaction occurring in untreated recipient mice.

introduction disease (GVHD) and long-lasting immunodeficiency are major complications of human allogeneic bone marrow transplantation (BMT).lJ GVHD is initiated by mature T cells of donor origin and results in the destruction of a number of target organs, including skin, gut and liver.3P Cyclosporine A (CsA) is a T cell-specific immunosuppressive agent that has been used to prevent organ graft rejection5 and GVHD after BM’I? Although CsA effectively down-regulates alloreactivity, GVHD has been shown to develop upon withdrawal of CsA therapy.’ Interestingly, an autoaggressive syn Graft-versus-host

Address for correspondence: J Annat, Departement Immunologie, Laboratoires Foumier SA, 50 me de Dijon, Daix 21121, France. 0 Arnold 1998

drome termed syngeneic GVHD can be obtained in rodents after lethal irradiation, syngeneic bone marrow reconstitution and CsA treatment.8 The appearance of autoreactive T cells seems to result from both thymic stroma injury caused by irradiation, and the effects of CsA on normal thymic positive and negative TCR repertoire selection.’ Although normal T cell repertoire negative selection was preserved in syngeneic Bm loss of normal thymic repertoire selection and persistence of peripheral autoreactive T cells have been reported in GVHD, following allogeneic BMT” These peripheral self-reactive T cells have been postulated to be responsible for the progression from acute to chronic GVI5D.l’ Moreover, overexpression of V$ gene segment transcripts in skin from patients transplanted with HLA-matched allogeneic bone marrow grafts was reported.‘&13 0966-3274(98)TI252OA

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Objectives LF084299 (‘Iksperimus) is a new immunosuppressive compound which has been shown to control alloreactivity and induce donor specific tolerance in an MHC mismatched rat cardiac transplant mode1.‘4v15Furthermore, we have previously demonstrated that administration of LF 08-0299 protect& irradiated recipient mice from lethal GVHD, related to either minor antigens alone or to the full H-2 barrierJ6 The purpose of the present study was to examine the effect of LF 08-0299 on normal thymic differentiation and T cell repertoire selection after irradiation and either syngeneic or semi-allogeneic BM’& to further investigate the immune status of recipient mice protected fkom GVHD. The presence of potent autoreactive peripheral T cells was determined by specific mAbs to the VP region of the TCR. Materials and methods Mice C57BU6.I (H-2b) and (C57B46 x DBA/2)Fl (B6D2Fl; H-2b’d) male mice, were purchased from CERJ (France). Animals were housed in microisolator filter-topped cages and used in experiments between 7 and 9 weeks of age. Total body irradiation and bone marrow transplantation B6D2Fl

recipient

mice were given a lethal dose of 12 Gy

by using a 6oCo source (1 Gy/min). Bone marrow cells were collected from femurs and tibias with RPM1 1640. B6D2Fl mice were reconstituted 16 h after irradiation, either by intravenous injection of 10’ bone marrow cells from B6D2Fl mice (syngeneic BMT) or 10’ cells from C57BIJ6 mice (allogeneic BMT). Recipient mice were monitored for clinical evidence of GVHD (weight loss, alopecia and skin erythema, diarrhoea, hunching). LF OS-OW9 admWbation ‘Resperimus, LF 08-0299 ([4-[(3+uninopropyl)amino]butyl] carbamic acid 2-[[6[(aminoiminomethyl)amino]hexyl]amino]-2oxo-methyl ester trihydrochloride) was synthesized at Fournier. LF 08-0299 was dissolved in 0.9% NaCl and administered daily for a period of 20 days by intraperitoneal injections at the dose of 1.25 mg/kg, previous defined as an effective dosage in the prevention of GVHD. “i6 ‘Ikeatment started 24 h after bone marrow transplantation. Immunofluorescence staining and flow cytometry

At various time points post-BMT, cells from lymph nodes (cervical, inguinal, axillary) were phenotyped. VP T cell repertoire was assessed by double-staining (2-5 x ld cells, 40 min at 4°C) using a FITC-conjugated anti-CD4 mAb (clone CT-CD4, Caltag) or a FITC-conjugated anti-CD8 mAb (clone CT-CD8a, Caltag), and PE-conjugated anti-VP mAbs (PharMingen). The following specific anti-VP mAbs were used in our study: VP3 (clone KJ25), Vfl5.1,5.2 (clone MR9-4), VP6

CD8

Figure 1 CD4 and CD8 expression on thymocytes of B6D2Fl mice treated with LF 0%02!39.The compound was administered for a period of 10 days. Thymocyte phenotypic analysis was performed on day 6, day 11 and 2 weeks after the end of treatment (day 2.5), and compared to nontreated mice (control). Representative results for four mice are shown as dot density plots. The percentage for each population is indicated in the appropriate box.

Tmmphnt Immum’ogy 1998; 6: 217-224

T cell repertoire expression in murine recipients of bone marmw transplant

(clone RR47), Vp.1, 8.2 (clone MR5-2), VP11 (clone RR315). Cells were analysed on an EPICS-PROFILE II (Coulter Electronics). CD4 and CD8 analyses were performed on viable lymphoid cells by gating on the basis of the forward and the side light scatter. VP histograms were derived from either positively stained CD4 or CD8 T cells. Thymocytes were phenotyped by double-staining using PE-conjugated anti-CD4 mAb (Caltag) and a FITC-conjugated anti-CD8 mAb (Caltag). Pa&o1ogic examiDation of timsues Mice were sacriliced and organs were placed in Bouin fixator, imbedded in paraffin, sectioned, mounted and stained with haematoxylin and eosin. Histologic analysis of cell necrosis and lymphocytic infiltrates was performed by a certified pathologist. Data analysis Group comparisons of VP phenotype data were made by analy-

sis of variance. Correlation between VP expression and cellularity was assessed by linear regression.

219

in B6D2Fl CD4 T cells was lower than in C57BU6 mice, likely to have resulted from normal deletion of V/38.1 T cells. LF 080299 therapy did not disturb ne@ive selection after synseneic BMT

thymic

To assess the influence of LF 08-0299 therapy on T cell development and thymic repertoire selection, B6D2Fl mice were lethally irradiated and transplanted with syngeneic bone marrow cells. The VP repertoire was analysed in peripheral lymph node T cells 20 days and 2 months post-B. Recipient mice retained the capacity to negatively select V$3, V/35.1, 5.2, and VP6 T cells in either CD4 T cell population (Figure 2) or CD8 T cells (not shown), comparable to unmanipulated naive B6D2Fl mice. The level of [email protected],8.2 expression was similar to naive B6D2Fl mice, but lower than naive C57BU6 mice in CD4 T cells. VP11 CD4 T cells were only partially deleted in irradiated recipient mice. The pattern of VP repertoire in CD4 (and CD8) T cell population from LF 08-0299-treated recipient mice was not altered, and was similar to control recipient mice treated with the vehicle, when analysed either at the end of treatment or 2 months post-BMT (Figure 2).

Results LF OS-0299 treatment blocks T cell differentiation in the thymus at an early step

We first assessed the effect of LF 08-0299 on the pattern of thymic differentiation. Naive B6D2Fl mice were sacrificed at various times post-LF 08-0299 treatment, and thymocytes examined by immunofluorescence double-staining for CD4 and CD8 receptors. Flow cytometric analysis revealed a progressive decrease in double-positive cells (DP), counterbalanced by an increased proportion of double-negative (DN) and singlepositive (SP) cells (Figure 1). Reduced numbers of thymic cells were found after LF 08-0299 treatment, indicating that the modified thymocyte subsets were mainly due to a major decrease in DP cells (-75% on day 6; -90% on day ll), with less severe reduction in the other compartments. These alterations were progressive (no effect on day 3) and fully reversible when analysed on day 25 (Figure 1). Histologic examination showed a progressive reduction in volwne and cellular content of the cortex, without evident change in the thymic medulla (data not shown). Normal thymic architecture was restored on day 25. No apoptotic cell death was detected in the thymus of LF 08-0299treated mice, using flow cytometric analysis after propidium staining and DNA fragmentation assay. In the same conditions, administration of dexamethasone rapidly induced apoptosis in more than 40% of thymocytes (data not shown). ‘ICR VP expression by C57BU6

and B6D2Fl lymph node T celis C57BU6 and B6D2Fl mice differ not only in their major and

minor histocompatibility antigens but also in their expression of Mls antigens. B6D2Fl mice express I-E class II molecule and bear endogenous retroviruses encoding several Mls antigens, and therefore delete T cells expressing particular VP rece tor molecules during T cell maturation in the thymus. ?,-20 C57BW6 mice do not express I-E class II molecule and therefore no VP population was deleted. B6D2Fl mice showed a complete deletion of Vp3, Vp5.1,5.2, Vp6, and VP11 T cells in CD4 T cell population (Figure 2). The same results were obtained in CD8 T cells, although VP11 appeared to be only partially deleted (not shown). The level of Vp8.1,8.2 expression Transplant Immunology 1998; 6: 217-224

Figure 2 Phenotypic analysis of VP repertoire in lymph node CD4 T cells, following syngeneic bone marrow transplantation. B6D2Fl recipients were lethally irradiated and transplanted with syngeneic bone marrow cells as described in ‘Materials and methods’. VP T cell repertoire was analysed at the end of LF 08-0299 treatment (day 20) and 2 months post-BMT (day 60). The VP pattern was analysed in LFW-0299 treated recipient mice (BMT + LFW-0299) and 0.9% NaCl treated recipient mice (Syngeneic BMT). VP pattern from naive C57BU6 and B6D2Fl mice was represented for normal level of expression and deletion. Data are expressed as mean percentage f SEM for six mice of each group, from two different experiments.

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Vf3 T cell pattern ie altered in Fl recipient mice receiving perentel bone merrow tmneplant

expanded Vj33 T cells in CD4 lymph node population. The frequency of Vi38.1,8.2 and V/j11 T cells was unchanged under LF 08-0299 therapy compared to control mice. In contrast, Vp5.1, 5.2 T cells were increased by LF 08-0299 administration in either CD4 T cell population (Figure 4) and CD8 T cells (not shown). The expansion of Vp.l,5.2 T cells was significant on day 20 and day 35 @ < 0.05), but not 2 months post-BMT

B6D2Fl mice lethally irradiated and reconstituted with parental bone marrow cells from C57BU6 mice are complete chimeras.16 The level of peripheral lymph node T cell reconstitution was significantly lower than for syngeneic BMT recipient mice at day 20 and even 2 months post-BMT (nble 1). However, one mouse showed a higher T cell reconstitution on day 60 and seemed to escape GVHR. The proportion of CD8 T cells was greatly increased compared to CD4 T cells in semiallogeneic recipient mice 20 days post-BMT, but returned to normal ratio 2 months pos&BMT Reconstitution of lymph node CD4 and CD8 T cells was not impaired under LF 08-0299 therapy and cellularity appeared higher, although non-significant, than in vehicle-treated recipients 2 months post-BMT (‘Ihble 1). In contrast to syngeneic BMT, parental BMT recipient mice showed altered VP repertoire pattern in peripheral T cells during GVHR. As shown in Figure 3, Vi33 and VW T cells were detected 20 days post-BMT in peripheral CD4 population, and were expanded at least three-fold compared to naive C57BL/6 mice. ‘Avo months post-BMT, VP3 and V/36 T cells persisted in either CD4 and CD8 populations, but expression level was similar to naive C57B46 mice. There was an inverse correlation (p < 0.05) between VP6 expression level in CD4 T cells, and the level of lymph node cellular reconstitution on day 20 and 2 months post-BMT The Vp5.1,5.2 and VP11 populations were not expanded in parental BMT recipient mice, but were expressed at a level comparable to the non-deleting C57BL/6 mice. In contrast, the expression level of Vfl8.1, 8.2 T cells in recipient mice of parental BMT was comparable to naive B6D2Fl in the CD4 and CD8 T cell populations, and lower than C57BW6 mice in CD4 T cells, similar to the pattern of expression in syngeneic BMT

Hietopathologic analysis No histopathologic abnormalities

characteristic of acute or chronic GVHD were observed 4 months post-BMT in skin, intestines or liver, although few periportal lymphocytic in% trates were shown in the latest (not shown). However, lymphoid tissues showed profound histopathologic alterations (Figure 5). Lymph node parenchyma was progressively substituted by an extensive fibrosis associated with depletion of lymphoid cells, and numerous histiocytic cells were observed in the sinus. An atrophy of the thymic medulla and a disappearance of the epithelial network were observed. In contrast, LF 08-0299-treated recipient mice showed normal lymph node parenchyma and lymphoid cell distribution, despite few histiocytic cells in the sinus. The histological aspect of the thymus was normal and no atrophy of the medulla was observed (Figure 5). Examination of skin, intestines and liver showed no histopathologic abnormalities, and no lymphocytic infiltrates were observed in liver. Diecuesion I3 08-0299is a new immunosuppressive

molecule currently under clinical investigation for the prevention of GVHD in unrelated BMT We have previously reported that LF 08-0299 was effective in the prevention of murine GVHD.16 lb further investigate the immune status of lethally irradiated recipients under LF OS-0299 therapy, we examined the effect of the cornpound on T cell repertoire selection. We first assessed the influence of in vivo administration on thymic differentiation. LF 08-0299 induced a progressive atrophy of the thymus predominantly affecting the cortex, which was not related to massive apoptosis induction. Phenotypic analysis rather showed that differentiation of thymocytes was partially blocked at an early step: the CD4_CD8- to CD4+CD8+ transition. Thus, the mechanism by which LF 08-0299 blocked thymic differentiation appears different from glucocorticoids~l and cyclosporine A which acts on more mature thymT” but is similar to those described for 15-deoxyspergualin. It has been demonstrated that CsA interfered with the deletion of autoreactive T cells during ontogeny in the thymus.g CsA

VP6 T cell expansion is decreaee dbyLFO8-0299 treatment in Fl recipients receiving parental BMT. In co&rest, VP5 T cells are overexpreseed under LF OS-0299 therapy

We investigated the influence of LF 08-0299 therapy on the pattern of VP repertoire in recipients of parental marrow transplant, 20 days, 35 days and 2 months post-BMT The expanded Vp6 T cells observed 20 days after transplantation was decreased under LF 08-0299 administration in CD4 lymph node T cell population (Figure 4) and in CD8 T cells (not shown). The decrease of Vp6 T cell expansion under LF 08-0299 treatment persisted on day 35 (p < 0.05) and day 60 post-BMT. In contrast, LF 08-0299 treatment did not influence the level of

lhble 1 Effect of LF 08-0299 on CD4 and CD8 lymph node T cell reconstitution of irradiated Fl recipient mice transplanted with parental bone marrow cells Day 20 post-BMT Group

CD4

Naive B6D2Fl Naive C57BU6 Syngeneic BMT Parental BMT Parental BMT + LFO8-0299

5.6 f 5.6 f 3.8 f 0.8 f 1.1 f

Day 60 post-BMT

CD8 1.1 1.1 0.6 O.l* 0.3

4.0 f 4.4 f 1.8 f 1.2 f 2.3 f

0.8 0.8 0.2 0.2 0.7

CD4

CD8

4.9 f 0.5 3.8 f 1.4 5.7 zt 0.6

2.5 f 0.3 1.5 f 0.5 1.7 * 0.2

B6D2Fl recipients were lethally irradiated and transplanted with syngeneic or parental bone marrow cells from C57BU6 mice as described in ‘Materials and methods’. Reconstitution of CD4 and CDS populations was analysed at the end of LF 08-0299 treatments (day 20) and 2 months post-BMT (day 60). Data are expressed as mean absolute cell number per lymph node (/16) f SEM for six mice ?f each group and from two different experiments. Statistical comparisons were made between syngeneic versus parental BMT and parental BMT versus LF 08-0299-treated parental BMT recipient mice. *p < 0.05 between syngeneic and parental BMT.

Tmnspht

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FilBpn 3 Phenotypic analysis of V8 repertoire in lymph node CD4 T cells, following parental bone marrow transplantation. B6D2Fl recipients were lethally irradiated and transplanted with parental bone marrow cells from CS7BIJ6 mice as described in ‘Materials and methods’. VP T cell repertoire was analysed 20 days (day 20) and 2 months post-BMT (day 60). The VP pattern was analysed in parental BMT recipient mice (Parental BMT) and naive C57BU6 and B6D2Fl mice. Data are expressed as mean percentage f SEM for six mice of each group, from two different experiments. Lymph nodes from several mice were pooled when low cellularity did not allow individual phenotyping.

administration has been reported to be associated with the development of an autoaggressive syndrome termed syngeneic GVHD in irradiated rodents receiving syngeneic donor hematopoietic grafts.24-26Recently, characterization of autoreactive T cell repertoire in syngeneic GVHD was reported.” Thus, we assessed the consequence of thymic differentiation blockade under LF 08-0299 therapy on T cell repertoire selection after lethal irradiation and syngeneic bone marrow reconstitution. Irradiated B6D2Fl recipient mice retained the capacity to negatively select V83, Vj35, VP6 and V88.1 T cells in CD4 and CD8 lymph node populations. These data suggest that Mls determinants are expressed and presented by cells within the thymus, so that negative selection of T cells can occur normally. Only the VP11 T cell population was partially deleted, perhaps due to a defect in a specific Mls expression or presentation. LF 08-0299 administration did not alter this pattern of VP repertoire expression in either CD4 or CD8 T cells, suggesting that the compound does not inhibit, at least in this strain, normal negative selection processes, despite a partial blockade of thymic differentiation at an early step. In contrast to syngeneic bone marrow transplant, loss of normal thymic repertoire selection and persistence of peripheral autoreactive T cells have been reported in GVHD.” As LF 08Transplant Immunology 1998; 6: 217-224

Figure 4 Vj3 repertoire expression in lymph node CD4 T cells of parental BMT recipient mice following LF 08-0299 therapy. B6D2Fl recipients were lethally irradiated and transplanted with parental bone marrow cells from C57BU6 mice as described in ‘Materials and methods’. V$ T cell repertoire was analysed at the end of LF 08-0299 treatment (day 20), 35 days (day 35) and 2 months post-BMT (day 60). The VP pattern was analysed in LFO8-0299 treated recipient mice (BMT + LFO8-0299) and 0.9% Nail treated recipient mice (Parental BMT). Data are expressed as mean percentage +- SEM for six mice of each group, from two different experiments. Lymph nodes from several mice were pooled when low cellularity did not allow individual phenotyping. *p < 0.05.

0299 has been shown to prevent lethal GVI-ID,16 we further investigated whether abnormal V8 T cell expression could be controlled under LF 08-0299 therapy, in the parental to Fl model. Recipient mice reconstituted with parental bone marrow cells did not show clinical evidence of acute GVI-Q however we found that a graft-versus-host reaction had occurred in lymph nodes with considerably lower cellularity than syngeneic BMT recipients. This lack of lymph node T cell reconstitution has previously been described in another donor recipient strain combination.28 In our parental to Fl BMT model, recipient mice showed abnormal V8 repertoire expression, with Vp3,

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Figure 5 Histopathologic analysis of lymphoid tissues. Examination of lymphoid tissues was performed 4 months post-parental BMT. Representativesections (magnifkationx250 and x25) of lymphnodes (A, B) and thymus(C, D) from LF 08-0299-treated recipientmice (B, D) and 0.9% NaCl-treated recipients(A, C).

Vp5.1,5.2, Vp6 and VP11 populations detected in lymph node CD4 and CD8 T cells. In contrast, Vp8.1, 8.2 expression level in CD4 T cells was similar to naive B6D2Fl mice and lower than

C57BIJ6 mice, suggesting that normal deletion of VP.1 T cells occurred. VP3 and VP T cells were considerably expanded in the CD4 T cell population 20 days post-BMT, but expression returned to levels similar to nondeleting mice 2 months postBMT. These cells could derive either from activation of donor mature T cells present in the bone marrow graft, or from transplanted hematopoietic precursors matured in a thymic microenvironment altered by GVHR. Abnormal thymic selection in mice undergoing GVHR has been well described, and aberrantly selected VP3 and VP6 T cells have been reported to maintain their responsiveness and to expand in vivo when transferred into sublethally irradiated recipients.‘op The presence of peripheral self-reactive T cells has been postulated to be responsible for the progression from acute to chronic GVHD.‘1*30 Several groups have reported that recognition of a host viral superantigen ma determine the manifestations of GVHD in murine models.26;1,32 L.F 08-0299 administration did not induce changes of Vp3, VfJ8.1,8.2 and VP11 T cell expression in peripheral lymph nodes of parental BMT recipient mice. However, the dramatic expansion of VpS T cells observed on day 20 post-BMT was selectively inhibited after LF 08-0299 therapy. Although expression of VP Transplant Immunology 1998; 6: 217-224

in control recipient mice decreased on day 35 and day 60 to reach a level similar to nondeleting mice, LF 08-0299-treated mice displayed persistent lower expression. In contrast, the Vg5.1,5.2 T cell population was increased in lymph nodes on day 20 and day 35 post-BMT. We have shown that lymph node T cell reconstitution appeared higher in LF 08-0299-treated recipients, suggesting a control of the GVHR. Histopathologic examination of lymph nodes 4 months post-BMT revealed normal histological structure, and showed that LF 08-0299 therapy effectively protected recipient mice from the GVHR. Atrophy of the thymic medulla and injury of medullary epithelial cells was also completely prevented by LF 08-0299 therapy. LF 08-0299 might exert its GVHR protective effect by preventing the activation and/or expansion of responsive T cells which bear Vp6 segment. VP subset overexpression has been recently described to be abolished in tolerant recipient mice protected from GVHD by transfer of donor cells immunized against the host?3 As VP6 T cell expansion was skewed toward an increase of VP5 T cells, it would be of interest to further assess the functional role of VP5 T cells in transfer experiments. We have previously shown that no lethal GVHD was induced in secondary recipients of host origin when spleen cells from recipients protected from GVHD were transferred.16 Whether it could be the result of active T cell suppression or anergy/ deletion of host-reactive T cells needs further investigation.

T cell repertoire expression

Acute and chronic GVIW have been .rqxxted to be due to different mechanisms stimulated by different MHC Ag?’ Thus, changes of the VP T cell pattern under LF 08-0299 therapy could be implicated as well in the control of chronic GVHD induction. Interestingly, we have reported16 that long-term surviving recipient mice protected from the GVHD by LF 08-0299 did not show histological signs of chronic GVHD or autoimmune-like syndrome as described for other immunosuppressive drugs.26*34 In summary, we have demonstrated that LF 08-0299 treatment did not disturb negative thymic selection in lethally irradiated recipient mice reconstituted with syngeneic bone marrow, despite a partial blockade of thymic differentiation under treatment. In the parental to Fl BMT model, administration of LF OS-0299 inhibited the expansion of VP6 T cells and induced an overexpression of VP5 T cells, without interfering with lymph node reconstitution in CD4 and CD8 T cells. Changes of the pattern of VP T cell expression under LF OS0299 treatment were associated with inhibition of the graft-versus-host reaction observed in lymph nodes and thymus. Further investigations are needed to better understand the mechanism of action of LF 08-0299 in the protection against GVHD. LF 08-0299 therapy may provide a novel strategy to prevent GVHD in humans undergoing unrelated BMT, and allow better lymphoid tissue reconstitution, preventing persistent immunosuppression observed in patients suffering from GVHD. Acknowledgements The authors wish to thank Agnes Martinien for her expert tech-

nical assistance in murine BMT and flow cytometric analysis, and Dr Patrick Roignot for histological analysis.

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recipients of fully mismatched donor marrow are protected from lethal graft-versus-host disease by in vivo administration of rapamycin but develop an autoimmune-like syndrome. J Irnmunol 1993; 151: 572Hl.