Immune reconstitution after bone marrow transplantation for ... - Nature

Bone Marrow Transplantation, (1999) 23, 451–457  1999 Stockton Press All rights reserved 0268–3369/99 $12.00 http://www.stockton-press.co.uk/bmt

Immune reconstitution after bone marrow transplantation for combined immunodeficiencies: down-modulation of Bcl-2 and high expression of CD95/Fas account for increased susceptibility to spontaneous and activation-induced lymphocyte cell death D Brugnoni1, P Airo`1, M Pennacchio2, G Carella1, A Malagoli1, AG Ugazio2, F Porta2 and R Cattaneo1 1

Servizio di Immunologia Clinica, 2Clinica Pediatrica, Spedali Civili and University of Brescia, Italy

Summary: We have studied the regeneration of T cell subsets and function after BMT in 21 children affected by combined immunodeficiency after BMT. In the first months, the striking predominance of CD4ⴙ cells displayed the primed CD45R0ⴙ phenotype and a high number of activated (HLA-DRⴙ) T cells were observed. Regeneration of naive CD4+CD45RA+ cells correlated with the recovery of proliferative responses to mitogens (r ⴝ 0.64, P ⬍ 0.001). Peripheral blood lymphocytes circulating after BMT undergo an increased process of in vitro cell death, resulting from two mechanisms: spontaneous apoptosis (SA), a consequence of defective production of IL-2 and down-regulation of Bcl-2 (P = 0.02 vs healthy controls), and high susceptibility to activation-induced cell death (AICD) after restimulation with mitogens. In accordance with the role of CD95/Fas in this latter process, we have observed a high level of CD95 expression (P ⬍ 0.001 vs healthy controls), correlated with AICD (P ⬍ 0.001) but not with SA, and decreasing with time after BMT (P ⬍ 0.001). Both SA and AICD levels correlated with the presence of activated T cells and decreased with the progressive recovery of T cell proliferative response. Therefore, the lymphocyte hyperactivated status might explain their susceptibility to apoptosis and contribute to the genesis of immunodeficiency that follows BMT. Keywords: bone marrow transplant; severe combined immunodeficiency; Fas; Bcl-2; apoptosis

ing susceptibility to infections even when T cell number is normal. In the present study we have investigated the role of apoptosis in the genesis of T cell dysfunction after BMT in a group of children affected by immunodeficiencies. Interestingly, it has been demonstrated that peripheral blood T cells regenerating after BMT are highly susceptible to in vitro spontaneous apoptosis (SA).3 This process results from inadequate stimulation and can be prevented by growth factors such as IL-2 and by activation of genes such as bcl-2.4–6 A different process of lymphocyte cell death results from repeated activation.5–7 In this process, termed activationinduced cell death (AICD), a key role is played by CD95/Fas, a molecule belonging to the tumour necrosis factor receptor superfamily, expressed by the large majority of CD45R0+ T cells, but by a much smaller number of CD45RA+ cells.8 It has been shown that cross-linking of CD95 causes cell death of sensitive cells9,10 and that the susceptibility to CD95-induced AICD is a function of the state of activation of CD45R0+ T cells.11,12 As T cells repopulating the peripheral blood after BMT display a characteristic CD45R0+ activated phenotype13–17 and defective production of IL-2,18 we have evaluated both SA and AICD and the expression of molecules involved in these processes (Bcl-2, CD95) in a group of patients transplanted for combined immunodeficiencies.

Materials and methods Patients

The majority of primary immunodeficiency disorders results from abnormalities in the differentiation or function of immunocompetent cells originating from haemopoietic stem cells. Stem cell transplantation, generally performed with BMT, therefore offers a therapeutic approach in these patients.1,2 Recovery of the T cell number after BMT is slow. The recovery of T cell function is even slower, caus-

Correspondence: Dr P Airo`, Immunologia Clinica, Spedali Civili Brescia, Piazza Spedali Civili, 1, 25123 Brescia, Italy Received 5 August 1998; accepted 20 October 1998

Twenty-one children who underwent BMT for primary combined immunodeficiencies were studied. The main clinical data are reported in Table 1. In vitro T cell depletion with Campath-1M was performed on all bone marrows obtained from HLA-haploidentical family members19 and in one patient (GRMA) transplanted from an unrelated donor mismatched for one locus, HLA-A. In three cases (CHAL, PEAN, GAMI), data presented concern a second BMT after failure of a previous one. Phenotypic analysis and proliferative response were evaluated every 1–2 months until +6 months, then every 6–12 months. Ten healthy children of comparable age (1–48 months) were

Role of Fas and Bcl-2 in lymphocyte apoptosis after BMT D Brugnoni et al

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Table 1

Patients’ features

Patient

Age/Sex

Diagnosis

Donor

Conditioning

GVHD a/c

GVHD prophylaxis

Acute GVHD treatment

Follow-up (months)

GRMA MOGI RICR SAAL SPCH SURO VAMI TEDA CAAN CHAL CHMI HOPE LUPA PEAN VELU DELY GAMI ABGI DUGA PEGI PADA

12/M 8/M 10/F 1/M 48/F 34/F 7/M 7/M 7/M 10/F 4/M 15/F 3/M 13/F 7/M 18/F 4/M 24/M 3/F 6/M 12/M

AR-SCID T⫺B⫹ Omenn’s syndrome Omenn’s syndrome XL-SCID T⫺B⫹ CID HLA II deficiency XL-SCID T⫺B⫹ SCID T⫺B⫺ Omenn’s syndrome AR-SCID T−B+ AR-SCID T−B+ SCID T−B+ XL-SCID T−B+ AR-SCID T−B+ XL-SCID T−B+ CID AR-SCID T−B+ CID CID XL-SCID T−B+ CID

1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 3 3 3 3

BuCy BuCy, VP16 BuCy ATG BuCy, Thiotepa BuCy BuCy BuCy BuCy, Thiotepa BuCy BuCy Cy, Thiotepa BuCy BuCy Cy, ATG BuCy, Thiotepa BuCy BuCy, Thiotepa ATG None BuCy

y/n y/n y/n n/n y/n y/n y/n y/n n/n n/n n/n n/y n/n y/y n/n y/n y/n y/n n/n n/n y/n

CsA CsA, ATG CsA CsA. MTX CsA CsA, MTX, ATG CsA CsA CsA, ATG CsA CsA CsA CsA CsA CsA CsA CsA CsA, ATG None None CsA

PDN, ATG PDN PDN, ATG None PDN, ATG PDN PDN PDN None None None None None PDN, ATG None PDN PDN PDN None None PDN

A ⫹50 A ⫹68 A ⫹48 A ⫹60 A ⫹29 D ⫹4 A ⫹41 A ⫹29 A ⫹24 A ⫹64 A ⫹35 D ⫹10 A ⫹75 D ⫹15 A ⫹65 A ⫹23 A ⫹19 A ⫹27 A ⫹52 A ⫹26 A ⫹24

Age is given in months. 1 = HLA-identical unrelated; 2 = HLA-haploidentical family donor; 3 = HLA-identical sibling; A = alive; D = dead.

studied as controls. Only data concerning the CD45 isoform expression and proliferative response in the groups of eight patients receiving BMT from unrelated donors and of nine patients receiving BMT from HLA-haploidentical family members have been described elsewhere.20,21

Peripheral blood mononuclear cell activation All experiments were performed in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, 1% glutamine and antibiotics. Proliferative responses were measured in triplicates of 200 ␮l containing 1 × 105 total PBMC using PHA, 1.2 mg/ml (Irvine Scientific, Santa Ana, CA, USA) or immobilised anti-CD3 mAb, 200 ng/ml (OKT3; Ortho, Raritan, NJ, USA). After 72 h of culture at 37°C, 5% CO2, the cells were pulsed overnight with 1 ␮Ci (3H)-thymidine. Apoptosis assay PBMC cultured with or without the stimulating agents in the conditions indicated above were also evaluated for apoptosis. In some experiments, recombinant human IL-2, 20 U/ml (Biosource International, Camarillo, CA, USA) was added. After culture, cells were washed and the percentage of apparently hypodiploid nuclei (less than 2 N DNA content) was determined with the method of Nicoletti et al22 slightly modified.23 Briefly, 1 × 106 lymphocytes were fixed in 1 ml of cold 70% ethanol at 4°C for 20 min. Cells were then washed, incubated at room temperature for 1 min with RNase (0.5 mg/ml; Boehringer, Mannheim, Germany), for 15 min with propidium iodide (100 ␮g/ml; Sigma, St Louis, MO, USA), and immediately analysed.

Lymphocyte surface phenotype analysis Evaluation of lymphocyte surface membrane expression was performed on EDTA-whole blood samples with the following mAbs: fluorescein-isothiocyanate (FITC)-conjugated OKT3/CD3, FITC or phycoerythrin (PE) OKT4/CD4, FITC or PE-OKT8/CD8, PE-OKDR (HLA-DR) from Ortho, PE-Leu45RO/CD45RO from Becton Dickinson (Mountain View, CA, USA), PE-2H4/CD45RA, from Coulter Immunology (Hialeah, FL, USA), and FITC-DX2 (CD95) from Pharmingen (San Diego, CA, USA). Samples were analysed with a flow cytometer equipped with an argon-ion laser (488 nm; Cytoron Absolute; Ortho). Analysis of intracellular Bcl-2 expression PBMC (5 × 105) were permeabilised using Permeafix (Ortho) for 40 min, prior to incubation with a rabbit polyclonal antibody to bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or with polyclonal non-immune rabbit IgG. After 30 min, cells were washed and incubated with FITC-conjugated goat anti-rabbit IgG antibody (Santa Cruz). Quantitation of positivity for Bcl-2 was performed using QuickCal beads (Flow Cytometry Standards, San Juan, PR) and results were expressed as specific MESF (molecule equivalents of soluble fluorochrome), after subtracting the values obtained for non-specific fluorescence (always ⬍2000 MESF).24 Statistical analysis Data are presented as the median (25th–75th percentile). Wilcoxon’s rank sum test was used for comparisons. Correlations were tested considering both linear and polynomial


models. Polynomial models were used when, comparing them by testing the reduction in error sums of squares against the residual error using standard analysis of variance techniques, a statistically significant improvement in the residual error was found.

explained by the progressive increase of the absolute number of CD45RA+ cells, whereas the absolute number of CD45R0+ remained fairly constant (Figure 2). A high proportion of activated T cells (CD3+ HLA-DR+) was also observed in the first months after BMT (months 1–4: 36% (26–61); P ⬍ 0.01 vs healthy controls), progressively decreasing with time (r = −0.57; P ⬍ 0.001).

Results Progressive rise of naive CD45RA+ and decrease of primed/activated CD45R0+ T cell subsets after BMT

100

The proliferative response to PHA was reduced in the first months after BMT, and increased progressively with time (linear regression analysis r = 0.54; P ⬍ 0.001; binomial regression analysis: r = 0.68; P ⬍ 0.001; Figure 3). Similar data were observed in anti-CD3-stimulated cultures (data not shown). Proliferative response significantly correlated with the proportion of CD4+CD45RA+ cells among lymphocytes (r = 0.64; P ⬍ 0.001; Figure 3), much more than with that of total CD4+ cells (r = 0.35; P = 0.003) but not with that of CD8+CD45RA+ cells (r = 0.06; P = NS). Moreover, it was inversely correlated with the proportion of CD3+HLA-DR+ cells (r = −0.50; P = 0.001) and of CD4+CD45R0+ cells (r = −0.34; P = 0.004). Taken together, % of CD45R0+ among CD4+ cells

% of CD45RA+ among CD4+ cells

In the first months after BMT, the striking predominance of CD4+ cells coexpressed the CD45R0 molecule, whereas CD4+CD45RA+ cells were initially rare. On the other hand, CD45RA+ and CD45R0+ subsets were almost equivalent among CD8+ cells (Figure 1). A progressive rise of the proportion of the CD45RA+ subset and, conversely, a decrease of the CD45R0+ subset were observed among CD4+ cells (linear regression analysis: r = 0.55; P ⬍ 0.001. In Figure 1 the result of binomial regression analysis is shown, since it better describes the fact that a plateau is reached by most patients; r = 0.72; P ⬍ 0.001). The same pattern, less marked, was observed also among CD8+ cells (linear regression analysis: r = 0.22; P = 0.02; binomial regression analysis: r = 0.42; P ⬍ 0.001). These changes were

The proliferative response is reduced after BMT and recovers in parallel with the regeneration of naive CD4+ T cells

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Figure 1 Progressive appearance of CD45RA cells and reciprocal decrease of the proportion of cells with CD45R0+ phenotype among CD4+ or CD8+ lymphocytes after BMT. Horizontal lines represent the median and the 25th–75th percentile of healthy controls.

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Figure 2 The absolute number of CD45RA+ (white) cells rises progressively after BMT whereas that of CD45R0+ cells remains fairly constant (black). Boxes represent 25th–75th percentile and horizontal lines within boxes represent the median.

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Figure 3 Progressive recovery of the proliferative response to PHA after BMT (left). Horizontal lines represent the median and the 25th–75th percentile of healthy controls. The recovery of proliferative response to PHA is correlated with the proportion of CD4+CD45RA+ cells among total lymphocytes (right).

these data suggest that the defective proliferative response is linked to the presence of activated T cells and recovers in parallel with the regeneration of naive CD4+ T cells. Analysis of variables which may influence the recovery of T cell number and function in these patients25 identified a significant role of the donor source and of the process of T cell depletion,20,21 whereas other factors (patient age; type of treatment post-transplant; presence of GVHD; type of immunodeficiency) did not have a major role. High levels of peripheral blood mononuclear cells SA and AICD can be observed after BMT We considered the possibility that the defect of lymphocyte proliferation was due to increased cell death during the culture, evaluating simultaneously the proliferative responses and tests for SA and AICD in 21 experiments performed on 12 patients (median time from BMT: 8 months; range 1–28). The numbers of cells dying in vitro either spontaneously in unstimulated culture (SA) or after stimulation (AICD) with PHA (Figure 4) or anti-CD3 (not shown) were higher in BMT recipients than in controls. Both the levels of SA and AICD inversely correlated with the proliferative response to PHA (r = −0.69; P = 0.001; r = −0.79; P ⬍ 0.001, respectively). The number of apoptotic cells also correlated with the proportion of activated peripheral blood T cells (CD3+ HLA-DR+) (SA: r = 0.80; P ⬍ 0.001; AICD: r = 0.68; P = 0.002). As the products of bcl-2 gene are known to modulate

the susceptibility of T cells to SA due to IL-2 starvation, intracytoplasmic levels of this protein were evaluated by flow cytometry in four patients (median time from BMT: 4 months; range 1–13). The intensity of lymphocyte staining for Bcl-2, expressed as MESF, after 48 h of unstimulated culture was significantly reduced in BMT recipients, as compared to six age-matched healthy controls (mean: 20780 MESF vs 40140; P ⬍ 0.02). Addition of rhIL-2 (20 U/ml) greatly reduced SA and down-modulation of Bcl-2 (Figure 5). Taken together, these data suggest that the raised number of spontaneously dying PBMC in patients after BMT may be partly accounted for by down-modulation of bcl-2 gene product expression caused by lack of IL-2. CD95/Fas expression was evaluated by flow cytometry at the time of the study of AICD (21 experiments), considering the role of the molecule in this phenomenon. CD95 expression by total peripheral blood lymphocytes, and in particular by CD4+ cells, from BMT recipients was higher than in controls (Figure 6a; P ⬍ 0.001) and decreased progressively (CD95 expression on CD4+ cells: r = −0.73 with time after BMT; P ⬍ 0.001). Moreover, CD95 expression was correlated with the levels of AICD (r = 0.69; P = 0.001), but not with that of SA (r = 0.22; P = NS) (Figure 6b). Discussion After BMT, a state of immunodeficiency persists for a long period. This cannot be attributed solely to the well known,


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Figure 4 Increased levels of spontaneous (left) and activation-induced (right) lymphocyte cell death in children after BMT. Boxes represent 25th–75th percentile and horizontal lines within boxes represent the median.

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Figure 5 (a) IL-2 reduces the rate of lymphocyte spontaneous cell death after 72 h of culture in children transplanted for immunodeficiencies. (b) The expression of Bcl-2 on PBMC culture in two representative children transplanted for immunodeficiencies and one healthy control after 48 h of culture in the absence (dark lines) or in the presence (light lines) of IL-2 (20 U/ml), which reduces the down-modulation of bcl-2.

quantitative defects of the CD4+ T cell subset.16,26,27 In fact, intrinsic qualitative defects of regenerating T cells have been demonstrated28 accounting for a prolonged impairment of proliferative responses to mitogens and nominal antigens. The phenotype of regenerating peripheral blood T cells in the early phases after BMT displays several features induced by prolonged stimulation, including expression of CD45R0, which is currently believed to require persistent antigenic stimulation,29 and of other activation markers such as HLA-DR.13–16 Conversely, the number of naive CD45RA+ T cells is low, and rises slowly after BMT.16,17 The ability to produce CD4 (but not CD8+) naive T cells is a function of patient age:16,27 in children T cell development after BMT may still partially follow a thymopoietic pathway.16,17,30 In contrast, defective regeneration of naive T cells after BMT in adults is attributed to thymus involution.30,31 The present study extends information on the T cell-acti-

vated phenotype in the early phases of regeneration after BMT, showing that they have the characteristics of cells prone to apoptotic processes, including reduced expression of bcl-2 gene product and high expression of CD95. We have therefore hypothesised that, as the result of this peculiar functional state of T lymphocytes, an excessive process of cell death accounts for their defective ability to proliferate in response to in vitro stimulation. Indeed, an increased rate of T cell SA after BMT has already been described, decreasing with time.3 We confirmed the increased rate of SA, and demonstrated also an increased susceptibility to AICD after re-stimulation in vitro with mitogens. Moreover, as expected according to our hypothesis, the progressive recovery of T cell proliferative response in vitro was correlated with the decrease of both SA and AICD. The process of in vitro cell death operating in patients after BMT results from two largely independent mechanisms. A first one is SA, caused by inadequate stimulation,


a % of positive cells

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Figure 6 (a) Increased expression of CD95/Fas by CD4 lymphocytes in children transplanted for combined immunodeficiencies. Proportion of CD4+Fas+ among total PBL (white) and of Fas+ among total CD4+ cells (black). (b) Fas expression by CD4+ cells is correlated with the levels of PHAinduced cell death (AICD), but not with spontaneous apoptosis.

as a consequence of down-regulation of Bcl-2, a protein that blocks growth factor deprivation-induced apoptosis of lymphocytes, but which seems to have no role in modulating CD95-induced AICD.5–7 Indeed, we have demonstrated a down-regulation of the expression of Bcl-2 in peripheral blood lymphocytes from BMT recipients that can be largely prevented by the addition of exogenous IL-2. This observation suggests that SA in these patients results from defective production of growth factors, such as IL-2, which leads to Bcl-2 down-modulation. In fact, it is known that PBMC from recipients of BMT produce less IL-2 in response to in vitro stimuli than do the same number of PBMC from healthy controls.18 A second mechanism is an increased susceptibility to AICD. It has been demonstrated that this process occurs mainly through a CD95/CD95 ligand-mediated mechanism,5–12 whereas CD95 does not have a role in the process of SA. Accordingly, we have observed that the high levels of CD95 expression observed on peripheral blood lymphocytes, and particularly on CD4+ cells, from transplanted children correlate with AICD, but not with SA. As a prolonged stimulation with mitogens plus IL-223 or multiple exposures to antigen6 are required to induce susceptibility to CD95-dependent AICD on mature T cells from normal individuals, these data confirm that a continuous antigenic stimulation is ongoing after BMT. This chronic immune activation may be responsible not only for CD95mediated AICD, but also for cell anergy due to starvation

from growth factors32 accounting for both mechanisms of cell death observed in our study. Therefore, the T cell hyper-activated status accounts for their susceptibility to apoptosis and impaired ability to mount a proliferative response, contributing to the genesis of immunodeficiency that follows BMT. Interestingly, we have demonstrated that a fast regeneration of normally functioning naive CD4+CD45RA+ T cells occurs after BMT from HLA-identical unrelated donors in children affected by (S)CID, leading to a full T cell reconstitution (including proliferative response) within 8 months.20 On the contrary, the generation of naive CD4+ cells is slow and impaired, not reaching normal levels even more than 1 year after BMT in children with (S)CID who received BMT after in vitro T cell depletion from HLAhaploidentical family donors.21 In this setting, hyperactivation of T cells, likely resulting from greater donor/recipient diversity, is more pronounced and prolonged21 and may contribute to the impairment of T cell reconstitution. Other factors may have a role, such as the very small number of donor T cells infused after T cell depletion27,33 in these patients. These observations are reflected clinically in a recent report of the European registry: overall survival of children with primary immunodeficiency after BMT from HLA-haploidentical family donor is much worse than after BMT from HLA-identical unrelated donors (which approaches that obtained with BMT from an HLA-identical sibling).34 In particular, the survival is very


poor in patients with defective T cell reconstitution, but good in those who achieved a full T cell reconstitution.34

18

Acknowledgements AM is the recipient of a grant from the Associazione Donatori Midollo Osseo.

19

References

20

1 Fischer A, Landais P, Freidrich W et al. European experience of bone marrow transplantation for SCID. Lancet 1990; 336: 850–854. 2 Fischer A, Landais P, Freidrich W et al. Bone marrow transplantation (BMT) in Europe for primary immunodeficiencies other than severe combined immunodeficiencies: a report from the European Group for BMT and the European Group for immunodeficiencies. Blood 1994; 83: 1149–1155. 3 Donnenberg AD, Margolick JB, Beltz LA et al. Apoptosis parallels lymphopoiesis in bone marrow transplantation and HIV disease. Res Immunol 1995; 146: 11–21. 4 Yang E, Korsmeyer SJ. Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood 1996; 88: 386–401. 5 Strasser A, Harris AW, Huang DCS et al. Bcl-2 and Fas/APO1 regulate distinct pathways to lymphocyte apoptosis. EMBO J 1996; 14: 6136–6147. 6 Van Parijs L, Ibramighov A, Abbas AK. The roles of costimulation and Fas in T cell apoptosis and peripheral tolerance. Immunity 1996; 4: 321–328. 7 Van Parijs L, Abbas AK. Role of Fas-mediated cell death in the regulation of immune responses. Curr Opin Immunol 1996; 8: 355–361. 8 Miyawaki T, Uehara T, Nibu R et al. Differential expression of apoptosis-related Fas antigen on lymphocyte subpopulations in human peripheral blood. J Immunol 1992; 149: 3753–3758. 9 Dhein J, Daniel PT, Trauth BC et al. Induction of apoptosis by monoclonal antibody anti-APO-1 class switch variants is dependent on cross-linking of APO-1 cell surface antigens. J Immunol 1992; 149: 3166–3173. 10 Suda T, Nagata S. Purification and characterization of the Fasligand that induces apoptosis. J Exp Med 1994; 179: 873–879. 11 Owen-Schaub LB, Yonehara S, Crump III WL, Grimm EA. DNA fragmentation and cell death are selectively triggered in activated human lymphocytes by Fas antigen engagement. Cell Immunol 1992; 140: 197–205. 12 Wesselborg S, Janssen O, Kabelitz D. Induction of activationdriven death (apoptosis) in activated but not resting peripheral blood T cells. J Immunol 1993; 150: 4338–4345. 13 Atkinson K. T cell subpopulations defined by monoclonal antibodies after human bone marrow transplantation. II. Activation and functional subsets of the helper-inducer and cytotoxic-suppressor subpopulations defined by two colour fluorescence flow cytometry. Bone Marrow Transplant 1986; 1: 121–132. 14 Leino L, Lilius EM, Nikoskeleainen J et al. The reappearance of 10 differentiation antigens on peripheral blood lymphocytes after allogeneic bone marrow transplantation. Bone Marrow Transplant 1991; 8: 339–344. 15 Gorla R, Airo` P, Ferremi-Leali P et al. Predominance of memory phenotype within CD4+ and CD8+ lymphocyte subsets after allogeneic BMT (letter). Bone Marrow Transplant 1993; 11: 346. 16 Storek J, Witherspoon RP, Storb R. T cell reconstitution after bone marrow transplantation into adult patients does not resemble T cell development in early life. Bone Marrow Transplant 1995; 16: 413–425. 17 Heitger A, Neu N, Kern A et al. Essential role of the thymus to

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