T cell immune reconstitution after allogeneic bone marrow transplantation in bare lymphocyte syndrome

T Cell Immune Reconstitution After Allogeneic Bone Marrow Transplantation in Bare Lymphocyte Syndrome Barbara C. Godthelp, Marja C. J. A. Van Eggermond, Maarten J. D. Van Tol, Jaak M. Vossen, and Peter J. van den Elsen ABSTRACT: To study the impact of an MHC class II-negative environment on T cell immune reconstitution, we have analyzed the phenotypical and functional characteristics of FACS-sorted cultured CD4⫹ and CD8⫹ T cells in two Bare Lymphocyte Syndrome (BLS) patients before and after allo-BMT. A similar analysis was performed in two MHC class II expressing pediatric leukemia patients after treatment with an allo-BMT who were included in our study as control. It was observed that CD4⫹ T cells displayed cytolytic alloreactivity in both BLS patients prior to and within the first year after allo-BMT, whereas such cells were absent at a later time-point, in the donors and pediatric leukemia controls. In addition, reduced MHC class II expression was observed in CD8⫹ T cells of both recipients early after allo-BMT, irrespective of the T

INTRODUCTION MHC class II deficiency, also referred to as Bare Lymphocyte Syndrome (BLS), is a rare primary immunodeficiency disease characterized by a complete absence of MHC class II molecules at the cell surface, often in conjunction with reduced MHC class I expression [1]. In BLS patients both cellular and humoral immune responses are severely impaired resulting in severe and recurrent infections mainly of the respiratory and gastrointestinal tract [2, 3]. The underlying genetic abnormality involves mutations in the genes encoding regulatory transacting factors: CIITA (complementation group A) [4] and subunits of the RFX complex, RFX-B/ From the Departments of Pediatrics (B.G., M.T., J.V.) and Immunohematology and Blood Transfusion (B.G., M.E., P.E.), Leiden University Medical Center, Leiden, The Netherlands. Address reprint requests to: Dr. Peter J. van den Elsen, Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Bldg. 1, E3-Q, Albinusdreef 2, P.O. Box 9600, 2300 RC Leiden, The Netherlands; Tel: ⫹31-71-5263831; Fax: ⫹31-71-5216751; E-Mail: [email protected]. Received March 24, 2000; accepted May 17, 2000. Human Immunology 61, 898 –907 (2000) © American Society for Histocompatibility and Immunogenetics, 2000 Published by Elsevier Science Inc.

cell chimerism pattern. Lack of endogenous MHC class II expression in BLS patients, therefore, results in aberrant T cell selection within the first year after allo-BMT, analogous to T cell selection before transplantation. These T cell selection processes seem to be normalized at a later time point after allo-BMT probably due to migration and integration of graft-derived MHC class II-positive antigen presenting cells to sites of T cell selection. Human Immunology 61, 898 –907 (2000). © American Society for Histocompatibility and Immunogenetics, 2000. Published by Elsevier Science Inc. KEYWORDS: CD4⫹ and CD8⫹ T lymphocytes; immunodeficiency diseases; MHC class II; bone marrow transplantation; cytotoxicity; allo-antigens

RFXANK [5, 6], RFX5 [7], and RFXAP [8], in complementation groups B, C, and D, respectively. The only curative treatment of this otherwise lethal immunodeficiency consists of allogeneic bone marrow transplantation (allo-BMT). However, the success rate of engraftment and immunological recovery in these patients is not yet comparable to that in other immunodeficiency conditions [9, 10]. The lack of MHC class II expression may present special problems with respect to T cell development and the generation of a fully competent immune repertoire in BLS patients. This is illustrated by reduced numbers of CD4⫹ T cells in the periphery [11], which display inverse skewing patterns of the T cell receptor (TCR) V gene segments and an altered amino acid composition of the TCR complementarity determining region 3 (CDR3) [11, 12] despite a diverse TCRAV and TCRBV gene family usage [11, 13]. These observations suggest that in the absence of endogenous MHC class II expression on thymic epithelial cells [11, 14], interactions of the TCR 0198-8859/00/$–see front matter PII S0198-8859(00)00156-7

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TABLE 1 Clinical characteristics of the MHC class II deficiency patients and allo-BMT outcome Patients Features CIDa type Affected gene Age at BMT Donor MHC class I/II-typing (patient⫽donor) Graftc Preparative regimend GVHD prophylaxise IVIGf Current status

Patient 1 Type III BLSb RFX-B (group B) 8 months HLA-identical sibling A2 A11 B18 B51 Cw5 DR7 DR11 DQ2 DQ7 2.5 ⫻ 108 Bu20 /Cy200 ⫹ ⫹ Alive and well ⬎ 2.5 years

Patient 2 Type III BLS RFX5 (group C) 23 months HLA-identical sibling A2 A3 B51 B50 Cw6 DR4 DR17 DQ2 DQ8 4.6 ⫻ 108 Bu20 /Cy200 ⫹ ⫹ Alive and well ⬎ 5 years

a

CID, combined immunodeficiency disease. Type III Bare Lymphocyte Syndrome (BLS): MHC class II-deficiency, with reduced levels of MHC class I expression. c The bone marrow graft depicted as nucleated cells/kg Body Weight (BW). d Bu20 ⫽ total dose of Busulfan in mg/kg BW. Cy200 ⫽ total dose of Cyclophosphamide in mg/kg BW. e GVHD prophylaxis consisted of Cyclosporin A 2 mg/kg/day IV for 1–2 months, 6 mg/kg/os 2– 6 months after allo-BMT and Methotrexate 10 mg/m2 on days ⫹1, ⫹3, ⫹6. f IVIG ⫽ intravenous immunoglobulins. Both children received IVIG after allo-BMT, Patient 1 for 1.5 months; Patient 2 was still on IVIG supplementation at the moment of this study. b

with alternative ligands such as MHC class I or CD1 may account for these observations [15, 16]. The poor success rate of allo-BMT in BLS patients may, therefore, also correlate with this lack of MHC class II expression. In particular, the thymopoietic or selection pathway of T cell reconstitution after allo-BMT, in which graft-derived donor precursor T cells expand after having been subjected to thymic [17, 18] and/or peripheral [19, 20] selection processes, is presumably severely impaired in these BLS recipients. Migration of graftderived mature T cells to the periphery [21–23] may, therefore, represent the only functional T cell immune reconstitution mechanism in BLS patients. These cells can be maintained in the periphery for over 10 –20 years [24] when appropriate TCR/MHC interactions occur [25]. In order to study the impact of a MHC class IIdeficient environment on T cell immune reconstitution, we have analyzed the phenotypical and functional properties of peripheral CD4⫹ and CD8⫹ T cells originating from two BLS patients before and after allo-BMT. METHODS Patients In 1995 and 1993, 2 unrelated BLS patients, Patient 1 (EBA, UPN 293) and Patient 2 (OSE, UPN 235), were transplanted in the Department of Pediatrics at the Lei-

den University Medical Center. They received a full bone marrow graft from their healthy HLA-identical sibling donors, Donor 1 [CBA, D(UPN293)] and Donor 2 [MSE, D(UPN235)] and showed successful engraftment with immunological recovery. The clinical characteristics of these BLS patients are presented in Table 1. Neither Patient 1 nor Patient 2 suffered from infectious complications after allo-BMT. Two leukemia patients treated with allo-BMT: Patient 3 [UPN 275] and Patient 4 [UPN285] were also analyzed and served as control [26]. They received a conditioning regimen consisting of a total dose of 120 mg/kg body weight (BW) cyclophosphamide, 4g/m2 cytosine arabinoside (Patient 3) or 700 mg/m2 etoposide (Patient 4) and a total dose of 7.5 Gy total body irradiation (TBI). They both received a full graft from HLA-matched sibling donors: Donor 3 [D(UPN275] and Donor 4 [D(UPN285]. Graft-versushost-disease (GvHD) prophylaxis consisted of cyclosporin A and methotrexate (similar to Patient 1 and Patient 2). Patient 4 suffered from acute GvHD (grade I), whereas GvHD was lacking in Patient 3. For the purpose of this study, blood samples were taken from both BLS recipients before allo-BMT, at 1 year and at 2 (Patient 1) or 4 (Patient 2) years after transplantation, from the leukemia patients at 1 year after BMT, and from all corresponding donors. The use of this human material has been approved by the Committee on Medical Ethics

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TABLE 2 Chimerisma patterns after allo-BMT Patient 1 Cell typeb

1 yr

T CD4⫹c CD8⫹ B NK MM

Patient 2 2 yrs

1 yr

4 yrs

D D D

D D D

D/R D/R R/d

D/r D/r D/r

R/D D D/r

nd nd nd

R/d R/d R/d

nd nd nd

a Chimerism determined via FACS-PCR-CA repeat analysis ⬇ at 1 year and at 2 or 4 years after allo-BMT. The predominant origin of the cell populations is given in capitals, either donor (D) or recipient (R), whereas a minor population (⬍10%) is indicated in undercast (d or r). If donor and recipient were present in equal amounts, both populations are given in capitals (R/D). b Cell types depicted are T lymphocytes (T), B lymphocytes (B), natural killer cells (NK) and myeloid and monocytic cells (MM), all populations were FACS-sorted from PBMC with appropriate monoclonal antibodies. c Similarly, CD4⫹ and CD8⫹ T cell subsets were obtained after FACS-sorting of PBMC after labeling with anti-CD4 and anti-CD8 mAbs. nd ⫽ not determined.

of the Leiden University Medical Center (Protocol: P254/96). Establishment and Characterization of Cultured CD4ⴙ and CD8ⴙ T cells Peripheral blood mononuclear cells (PBMC) of the donors and recipients of the allo-BMT were separated over a Ficoll-Isopaque gradient. Subsequently, CD4⫹ and CD8⫹ T cell subsets were FACS-sorted and taken into culture with phytohaemagglutinin (PHA), rIL-2 and irradiated feeders (PBMC from a buffycoat pool of 5

random donors) as described previously [27, 28]. Between day 10 and 14, mitogen stimulated T cells were either tested for their cytotoxic and proliferative capacity or for their MHC class II cell surface expression. MHC class II expression was investigated by staining with fluorescein-conjugated anti-HLA-DR mAb (Becton Dickinson) and subsequent analysis on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA). Chimerism patterns were regularly evaluated using PCR of CA-repeats on FACS-sorted cell populations [29, 30] i.e., T lymphocytes (CD3⫹), B lymphocytes (CD19⫹CD20⫹), natural killer cells (CD3⫺CD16⫹) and myeloid and monocytic cells (CD14⫹CD33⫹) (Table 2). Standard immunophenotypical analysis of PBMC [26, 30] was performed at regular intervals before and after BMT (Table 3). Cytotoxicity Assay MHC-class I and class II matched and mismatched (⫽third party) Epstein Barr virus (EBV) transformed B lymphoblastoid cell lines (BLCL) were labeled with Na251CrO4 (50 ␮Ci/1 ⫻ 106 B cells, New England Nuclear, Boston, MA, USA) for 1 h at 37°C. After 3 washes in HBSS, 2 ⫻ 103 labeled target cells were co-incubated with 8 ⫻ 104 cultured (see previous text) CD4⫹ or CD8⫹ effector T cells and incubated for 4 h at 37°C. Target cells were either incubated with medium alone or with 2% Triton X-100 to determine the spontaneous and the maximal Na251CrO4 release, respectively. The 51Cr content of the supernatant was measured by gamma counting. The percentage specific lysis was

TABLE 3 Immunophenotypical analysisa before and after allo-BMT CD4⫹ (⫻103/␮l)

Individual (age)

CD4⫹CD45RA⫹ CD4⫹CD45RO⫹ (⫻103/␮l) (⫻103/␮l)

CD8⫹ (⫻103/␮l)

CD8⫹CD45RA⫹ CD8⫹CD45RO⫹ (⫻103/␮l) (⫻103/␮l)

Patient 1 pre-BMT (1 yr) post-BMT 1 yr (2 yrs) post-BMT 2 yrs (3 yrs)

0.95 (34%) 1.24 (20%) 0.62 (19%)

0.58 (60%) 0.53 (43%) 0.20 (32%)

0.33 (34%) 0.81 (65%) 0.44 (70%)

0.36 (13%) 2.29 (37%) 1.18 (36%)

0.24 (66%) 1.24 (54%) 0.67 (57%)

0.12 (32%) 1.51 (66%) 0.72 (61%)

Patient 2 pre-BMT (23 mo) post-BMT 1 yr (3 yrs) post-BMT 2 yrs (4 yrs)

0.13 (12%) 0.11 (10%) 0.38 (10%)

0.04 (34%) 0.03 (27%) 0.12 (30%)

0.09 (70%) 0.08 (71%) 0.27 (72%)

0.55 (50%) 0.33 (29%) 1.44 (37%)

0.45 (81%) 0.30 (85%) 1.09 (78%)

0.11 (21%) 0.05 (14%) 0.44 (31%)

0.32 (40%) 0.31 (82%)

0.51 (63%) 0.07 (19%)

Referenceb

0–1 yr 1–6 yrs

Donor 1 Donor 2

(7 yrs) (12 yrs)

Reference

7–17 yrs

2.2 (1.7–2.8) 1.6 (1.0–1.8) 1.05 (30%) 0.56 (37%) 0.8 (0.7–1.1)

1.8 (1.1–2.5) 1.1 (0.7–1.4) 0.50 (48%) 0.34 (60%) 0.5 (0.4–0.7)

0.9 (0.8–1.2) 0.9 (0.8–1.5) 0.61 (60%) 0.20 (35%)

0.81 (23%) 0.38 (25%) 0.8 (0.6–0.9)

a The percentage of cells was determined after staining with appropriate mAbs and analysis on a FACscan. For all markers both absolute numbers as well as percentages (in parentheses) are given. b Age-matched reference values according to Erkeller-Yuksel et al. (57), are given as median, with ranges from 25 to 95 percentiles in parentheses. Reference values for CD4⫹CD45RO⫹ and for CD8⫹CD45RA⫹ and CD8⫹CD45RO⫹ are not available.

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calculated using the following formula: % specific lysis ⫽ (experimental release ⫺ spontaneous release)/(maximal release ⫺ spontaneous release) ⫻ 100%. Each experiment was performed in triplicate and repeated at least two times to ensure reproducibility. Cytotoxicity inhibition assays were performed by pre-incubating Na251CrO4 labeled targets with anti-MHC class I mAb (W6/32 [31]; 1:200) or the MHC class II mAb (PdV5.2 [32]; 1:200) for 30 min at 37°C prior to co-incubation with T cells. The antibodies remained present during the entire assay. Mixed Lymphocyte Reaction The proliferative capacity of the cultured CD4⫹ T cells was tested in a mixed lymphocyte reaction and measured by a standard 3H-thymidine incorporation assay. In triplicate, 1 ⫻ 106/ml stimulator BLCL (MHC matched/ mismatched) were irradiated (5000 Rad) and mixed with 1 ⫻ 106/ml CD4⫹ T cells from either donor or alloBMT recipient and incubated for 4 days at 37°C in humidified air with 5% CO2. Sixteen hours before harvesting 0.5 ␮Ci 3H-thymidine was added to each well and thymidine incorporation into DNA was measured using liquid scintillation counting. The proliferative capacity of cultured CD4⫹ T cells was performed in triplicate and analyzed twice to ensure reproducibility.

FIGURE 1 FACS analysis of MHC class II cell surface expression after allo-BMT. CD4⫹ and CD8⫹ T cells were cultured in vitro by stimulation with PHA and rIl-2 in the presence of irradiated feeders (see Materials and Methods). The percentage MHC class II (HLA-DR) positive CD4⫹ and CD8⫹ T cells at several time-points after allo-BMT was determined after labeling with a FITC conjungated anti-MHC class II mAb (BD ␣-HLA-DR). Ig isotype-FITC was used as negative control that did not exceed 101 fluorescence intensity. The fluorescence intensity of the DR-FITC stained cells on average was between 101 and 103. Shown are the results of BLS patients, Patient 1 (P1) and 2 (P2), at 1 and at 2 (P1) or 4 (P2) years after allo-BMT and of their corresponding donors (D1, D2). Leukemia patients, Patient 3 (P3) and 4 (P4) at 1 year after allo-BMT and their donors (D3, D4) were also analyzed.

RESULTS Hematopoietic Cell Lineage Chimerism Patterns After Allo-BMT Chimerism analysis at 1 year after allo-BMT revealed that in Patient 1 the majority of the hematopoietic cell populations was of donor-origin, whereas in Patient 2 most cells were of recipient origin (Table 2). However, the T cell chimerism pattern in Patient 2 changed to predominantly donor T cells at 4 years post-BMT (Table 2). Similar analyses in leukemia patients also revealed varying chimerism patterns in all tested hematopoietic lineages ranging from a complete donor-chimerism to a stable mixed chimerism [26, 33] (results not shown).

3). In both patients the CD8⫹ T cells were primarily of the CD45RA⫹ phenotype. After allo-BMT, 27 to 43% of the CD4⫹ T cell population was of the CD45RA⫹ phenotype in both patients. In Patient 2 the absolute number of CD4⫹CD45RA⫹ increased in time, whereas in Patient 1 the absolute number of naive CD4⫹ T cells slightly decreased. In the CD8⫹ T cell subset 54 to 85% of the cells was of the CD45RA⫹ phenotype at 1 to 2 years after allo-BMT. Immunophenotypical analysis in the leukemia patients (Patient 3 and 4) revealed similar results with a slow recovery of CD45RA⫹ CD4⫹ T cells, i.e., below reference values at 1 year after BMT, and a relatively quick recovery of CD45RA⫹ CD8⫹ T cells [26] (results not shown).

Immunophenotypical Analysis Before and After Allo-BMT Analysis of lymphocyte subsets revealed that both BLS patients had low numbers of CD4⫹ and CD8⫹ T cells prior to allo-BMT when compared with age-matched controls. After allo-BMT, low numbers of CD4⫹ T cells were observed, whereas CD8⫹ T cell numbers slowly increased and reached normal absolute values within 2 years after transplantation (Table 3). Analysis of the CD45RA expression revealed that approximately 60% of the CD4⫹ T cells in Patient 1 and 34% in Patient 2 were of the CD45RA⫹ phenotype prior to allo-BMT (Table

MHC Expression Patterns of T Cells After Allo-BMT The MHC expression patterns of CD4⫹ and CD8⫹ T cell subsets derived from both BLS patients and donors were analyzed after mitogenic stimulation (see Methods section). Before allo-BMT, T cells of BLS patients lack MHC class II (HLA-DR) expression as described previously [1, 34]. However, it should be noted that in Patient 1 less than 5% of the monocytes (CD13⫹) and B cells (CD20⫹) showed some residual MHC class II expression before allo-BMT (results not shown). Reduced expression of MHC class II (HLA-DR) was noted in

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FIGURE 2 Cytotoxic allo-reactivity of CD4⫹ and CD8⫹ T cells before and after allo-BMT. Donor-derived BLCL, thirdparty BLCL and NK cell target K562 were labeled and used as targets in a 4 h 51Cr release assay. (A) CD4⫹ and CD8⫹ T cells obtained from Patient 1 before allo-BMT and at 1 year (t ⫽ 1 year) after BMT, and from Donor 1 were used as effectors. (B) Comparison of allo-reactivity in both T cell subsets in Patient 1 at 1 year and at 2 years (t ⫽ 2 years) after allo-BMT. (C) T cells obtained from Patient 2 prior to allo-BMT and 1 year after allo-BMT (t ⫽ 1 year), and from Donor 2 were used as effectors. (D) Comparison of allo-reactivity of Patient 2-derived T cells at 1 year and at 4 years (t ⫽ 4 years) after BMT. Depicted is the percentage killing of labeled target cells at an effector/target ratio of 40:1. P1 ⫽ Patient 1, D1 ⫽ Donor 1, P2 ⫽ Patient 2, D2 ⫽ Donor 2, TP ⫽ third party.

particular within the CD8⫹ T cell subset and to a lesser extent also in the CD4⫹ T cells in both patients at 1 year after allo-BMT (Fig. 1). However, this reduced expression was normalized at two (Patient 1) or 4 years (Patient 2) after transplantation (Fig. 1). This reduced MHC class II expression was not found in allo-BMT recipients treated for leukemia at 1 year after allo-BMT (Fig 1). Functional Properties of CD4ⴙ and CD8ⴙ T Cell Subsets Cytolytic capacity. Cultured peripheral CD4⫹ T cells of Patient 1 exhibited cytolytic allo-reactivity with 29 to

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38% target cell lysis against third-party BLCL both before and at 1 year after allo-BMT (Fig. 2A). The erythroblastoid cell line K562 and MHC-matched BLCL of Donor 1, were not lysed by these effector T cells. However, this cytolytic activity of CD4⫹ T cells was lost at 2 years post-BMT (Fig. 2B). As expected, CD8⫹ T cells of Patient 1 showed similar cytolytic activity before and at 1 year and 2 years after allo-BMT with 25 to 35% target cell lysis. Similar results were obtained with CD4⫹ and CD8⫹ T cells of Patient 2 (Figs. 2C and 2D). Analysis of donor T cells and of T cells from leukemia patients (Patients 3 and 4) treated with an allo-BMT only revealed efficient cytolytic allo-activity of the CD8⫹ T cell subset (Figs. 2A and 2C and results not shown). The cytolytic activity of the BLS-derived CD4⫹ T cells could be blocked by an anti-MHC class I mAb as well as an anti-MHC class II mAb prior to as well as after allo-BMT (Figs. 3A and 3B). The cytolytic activity of the CD8⫹ T cells of both BM-donors were predominantly MHC class I-restricted as shown in Figs. 3A and 3B. A similar inhibition of cytolysis was noted with the BLSderived CD8⫹ T cells. In addition the CD8⫹ T cells of P1 displayed considerable inhibition of cytolysis also with the anti MHC class II antibody (Fig. 3A). The CD4⫹ and CD8⫹ T cells of BLS recipient and

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FIGURE 3 Inhibition of cytolytic response of CD4⫹ and CD8⫹ cells with anti-MHC class I and/or anti-class II mAbs. (A) T cells of Donor 1 and of Patient 1 obtained before and at 1 year after allo-BMT, respectively, were used as effectors and third-party BLCL as targets, after preincubation of targets with either anti-MHC class I mAb (W6/32) or anti-MHC class II mAb (PdV5.2) at an effector/target ratio of 40:1. Depicted is the percentage inhibition of target cell lysis after addition of the mAb. (B) T cells of Donor 2 and of Patient 2 obtained before and at 1 year after allo-BMT, respectively, as effectors and third-party BLCL as targets. P1 ⫽ patient 1, P2 ⫽ Patient 2, D1 ⫽ Donor 1, D2 ⫽ Donor 2.

donor showed Fas ligand mRNA expression as assessed via RT-PCR and Fas (CD95) cell surface expression (results not shown). Intracellular perforin expression was high in CD8⫹ T cells and intermediate in CD4⫹ T cells as was the cell surface expression of the cytolytic T cell marker p38 [35] (results not shown). However, no striking differences were observed between the cytolytic CD4⫹ T cell lines and the non-cytolytic ones of patients and donors. Proliferative Capacity Normal proliferative responses to allo-antigens were observed in CD4⫹ T cells of Patient 1 both before and at 1 year after BMT (Fig. 4). Donor 2- and third partyderived BLCL were recognized very well (16 – 41 ⫻ 103 cpm), whereas MHC-matched Donor 1-derived B cells were hardly recognized (3 ⫻ 103 cpm). In contrast, in CD4⫹ T cells of Patient 2 a low proliferative response (4 – 6 ⫻ 103 cpm) was observed before allo-BMT, irrespective of the stimulator cells used (Fig. 4). Similar analysis in Patient 2 demonstrated a normal proliferative response to allo-antigens at one year after allo-BMT. Third party-derived BLCL were recognized very well (23–27 ⫻ 103 cpm), whereas Donor 2-derived BLCL were hardly recognized (Fig. 4).

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DISCUSSION In the present study, we have evaluated the impact of MHC class II deficiency on T cell immune recovery in two BLS patients. The immunophenotypical analyses on freshly isolated PBMC revealed a slow recovery of CD4 ⫹ CD45RA ⫹ T cells and a relative quick recovery of CD8 ⫹ CD45RA ⫹ T cells in both BLS patients. This is in concordance with data obtained from leukemia patients after allo-BMT [26, 33]. The expression of CD45RA⫹ on a subset of the CD4⫹ T cells, therefore, suggests that these T cells could represent recent thymic emigrants that have been subjected to T cell selection processes in the microenvironment of the thymus. We have shown that in the absence of endogenous MHC class II expression, recipient-derived T cells of FIGURE 4 Proliferative capacities of CD4⫹ T cells before and after allo-BMT. A 3H-thymidine incorporation assay was performed with MHC class I and II matched and mis-matched irradiated BLCL as stimulators and CD4⫹ T cells from both donor/recipient couples as effectors, at an effector/stimulator ratio of 1:1. Depicted is the average 3H-thymidine incorporation of a triplicate assay in counts per minute (cpm) for each effector-stimulator combination. P1 ⫽ Patient 1, D1 ⫽ Donor 1, P2 ⫽ Patient 2, D2 ⫽ Donor 2, TP ⫽ third party.

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donor origin expressed reduced levels of MHC class II at 1 year post-BMT after in vitro culture. This was particularly noted on cultured CD8⫹ T cells and to a lesser extend also on cultured CD4⫹ T cells of both allo-BMT recipients, irrespective of the T cell chimerism [26, 34]. The T cells of the donor and the T cells of the leukemia patients after allo-BMT showed normal MHC class II expression. Since these mitogen stimulated cells expressed the activation associated markers CD45RO and CD25 [34] the aberrant MHC class II expression does not represent a general activation defect in these cells. Therefore, the exact cause of the aberrant MHC class II expression in the BLS recipients, in particular on the donor-derived CD8⫹ T cells, remains to be investigated. However, it does not seem to be related to the BMT procedure, because leukemia patients treated with alloBMT show normal MHC class II expression on T cells. A similar analysis at a later time-point after allo-BMT revealed normalization of the MHC class II expression on the CD8⫹ and the CD4⫹ T cells in both BLS patients, which in Patient 2 was accompanied by a change in T cell chimerism. In addition, we have shown that CD4⫹ T cells have acquired cytolytic functions in the absence of endogenous MHC class II expression. This activity was noted prior to and at 1 year after allo-BMT, and is presumably resulting from lack of MHC class II-mediated T cell selection in the thymus as a consequence of the defect in transcription factors critical to MHC class II expression. The observed cytolytic activity was specific, because only third-party MHC class I and class II mismatched targets were killed. Since cytolytic activity against sibling donor B cells was lacking, and since blocking of cytolysis of third-party B cells could be obtained with anti-MHC class I mAbs, these observations infer that at least part of these cytolytic CD4⫹ T cells have been selected via MHC class I in the absence of endogenous MHC class II [11, 14]. The fact that the cytolytic activity of the CD8⫹ T cells of P1 was also blocked with an anti-MHC class II antibody perhaps is resulting from absence of MHC class II-mediated negative selection processes in this patient. The loss of cytolytic CD4⫹ T cells that was observed at a later time-point after allo-BMT is most probably the result of normalization of precursor T cell selection similar to the situation in leukemia recipients. These cytolytic CD4⫹ T cell lines expressed Fas ligand mRNA and showed weak intracellular perforin expression, revealing that both killing mechanisms could have been used by these T cell lines. This is in agreement with previously published data [36, 37]. Interestingly, the existence of cytolytic CD4⫹ T cells has been reported before [36, 38, 39], but these CD4⫹ T cells were MHC class II-restricted and are thought to have an immunoregulatory function [40]. MHC class I-restricted CD4⫹

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cytotoxic T cells, as we have found, is a less frequent observation [41, 42]. The proliferative capacity of CD4⫹ T cells was different when comparing the two BLS patients. Normal proliferative responses against allo-antigens were found in Patient 1 prior to allo-BMT which agrees with previous publications [43, 44], whereas CD4⫹ T cells of Patient 2 lacked such activity. These observations could indicate that residual MHC class II expression in the thymus or on monocytes and/or B cells of Patient 1 (RFX-B deficient) may have contributed to toleratization of peripheral T cells to self-MHC prior to allo-BMT. Such MHC class II-positive tolerizing cells were lacking in Patient 2 (RFX5 deficient) [11, 14] resulting in low levels of recognition of self-MHC as well as non-self MHC. Both patients display normal proliferative responses to allo-antigens after allo-BMT revealing proper self–nonself discrimination most likely due to interaction with MHC class II-positive donor-derived cells. The mechanism by which peripheral T cells have been selected, if they are selected at all, in an environment lacking endogenous MHC class II is still unclear. During normal T cell development, bone marrow derived T cell precursors are subjected to positive and negative selection processes in the thymus upon interaction with MHC class I and MHC class II molecules expressed on thymic epithelial and dendritic cells [45, 46]. Mouse models with variable MHC expression have shown that TCR/ MHC interactions are required for the initial stages of positive selection [47] and that in MHC-negative hosts positive selection could be restored upon introduction of MHC-positive thymic epithelial cells or fibroblasts [48, 49]. It was originally thought that bone marrow derived cells were not involved in T cell selection processes [50]. However, a clear role for these bone marrow derived MHC expressing cells in T cell selection has been established recently [51]. It explains the change in function and phenotype of the CD4⫹ T cells in these BLS patients at a later timepoint after allo-BMT. Little is known about the MHC class II expression in the thymus of BLS patients of the various complementation groups. To date, only patients with a defect in RFX5 (group C) [52] have been analyzed and these thymi were shown to be completely devoid of MHC class II molecules [11, 14]. This is in contrast with data obtained from CIITA⫺/⫺ and RFX5⫺/⫺ knock-out mice, which have residual MHC class II expression in the thymus [53–55] and emphasizes the difficulty of comparing mouse-models with the actual human disease. In conclusion, due to the lack of endogenous MHC class II expression in the thymus and periphery of BLS patients [11, 14], donor-derived precursor T cells are presumably not properly selected into CD4⫹ and CD8⫹ single-positive T cells early after allo-BMT. However,

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these aberrant T cell selection processes might be partially restored by proper migration and integration of MHC class II-positive dendritic cells or monocytes [56] into the thymus or into alternative T cell selection sites like gut or liver at a later time-point after allo-BMT [19, 20]. 10. ACKNOWLEDGMENTS

We are indebted to L. Wilson for expert technical assistance, and to Drs I.I.N. Doxiadis and G.M.Th. Schreuder for critically reading the manuscript. This research was supported in part by the J.A. Cohen Institute for Radiopathology and Radiation Protection (IRS).

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