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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 348-358
By
From the Departments of Pediatrics and Immunohematology and Blood
Bank, Leiden University Medical Center, Leiden, The Netherlands; the
Immunology Unit, Haceteppe University Childrens Hospital, Ankara,
Turkey; and the Heilig Hart Hospital Tienen, Tienen,
Belgium.
To study the effects of major histocompatibility complex (MHC) class
II expression on T-cell development, we have investigated T-cell immune
reconstitution in two MHC class II-deficiency patients after
allogeneic bone marrow transplantation (allo-BMT). Our study showed
that the induction of MHC class II antigen expression on BM
graft-derived T cells in these allo-BMT recipients was hampered upon
T-cell activation. This reduction was most striking in the CD8+ T-cell subset. Furthermore, the peripheral T-cell
receptor (TCR) repertoire in these graft-derived MHC class
II-expressing CD4+ and in the CD8+ T-cell
fractions was found to be restricted on the basis of TCR complementarity determining region 3 (CDR3) size profiles.
Interestingly, the T-cell immune response to tetanus toxoid (TT) was
found to be comparable to that of the donor. However, when comparing
recipient-derived TT-specific T cells with donor-derived T cells,
differences were observed in TCR gene segment usage and in the
hydropathicity index of the CDR3 regions. Together, these results
reveal the impact of an environment lacking endogenous MHC class II on
the development of the T-cell immune repertoire after allo-BMT.
IN ORDER TO EVALUATE the effects of an
environment devoid of endogenous major histocompatibility complex (MHC)
class II antigen expression on T-cell immune reconstitution, we have performed a detailed analysis of the overall peripheral T-cell receptor
(TCR) repertoire and the tetanus toxoid (TT) specific TCR repertoire in
two MHC class II-deficiency patients after allogeneic bone marrow
transplantation (allo-BMT). We have generated CD4+ and
CD8+ T-cell lines and studied MHC expression patterns and
TCR complementarity determining region 3 (CDR3) size profiles for the
analysis of the overall T-cell repertoire. TT-specific T-cell lines
were used for the analysis of the antigen-specific T-cell repertoire
upon analysis of TCR gene segment usage, and hydropathicity index of the CDR3 region.
MHC class II deficiency, also referred to as bare lymphocyte syndrome
(BLS), is a rare immunodeficiency disease, inherited in an autosomal
recessive fashion. It is characterized by defective expression of MHC
class II antigens on all cell types, in conjunction with a varying
degree of MHC class I antigen expression, resulting in severely
impaired cellular and humoral immune responses upon antigenic
challenge. As a consequence, these patients are extremely susceptible
to viral, bacterial, and fungal infections.1-4 The underlying genetic abnormality involves mutations in genes that encode
transcription factors controlling MHC class II expression: CIITA (group
A)5 and subunits of the RFX complex, RFXANK (group B),6 RFX5 (group C),7 and RFXAP (group
D).8,9 Currently, the treatment of choice for this
otherwise lethal immunodeficiency is allo-BMT. However, the success
rate of engraftment and immunological recovery in BLS patients is lower
than in patients with other immunodeficiency syndromes, especially
following allo-BMT with BM grafts that are not
HLA-identical.10-12
During normal T-cell development, BM-derived precursor T cells home to
the thymus, where they are subjected to positive and negative selection
processes upon interaction with MHC class I and II molecules expressed
on thymic epithelial and dendritic cells of the cortex and medulla,
respectively.13-17 These selection processes within the
thymic microenvironment result in a peripheral pool of T cells that
does not respond to self-peptides but is able to recognize foreign
peptides in the context of self-MHC. Within the thymic
microenvironment, MHC class II-mediated interactions result mostly in
CD4+ T-cell development, whereas MHC class I-mediated
interactions result in CD8+ T-cell
development.18 Despite a general lack of endogenous MHC
class II expression19,20 in the thymic microenvironment, a
small number of CD4+ T cells is still found in the
peripheral compartments of BLS patients.20,21 These
observations imply that alternative ligands, such as MHC class I
antigens, may have mediated the development of
CD4+CD8+ thymocytes into CD4+ T
cells.22 On the other hand, it cannot be excluded that
these CD4+ T cells have reached the circulation without any
selection. Interestingly, these BLS patient-derived CD4+ T
cells show diverse TCRAV and TCRBV gene family
usage20,23,24 with inverse TCRAV/TCRBV skewing
patterns.20,25 Moreover, the lack of MHC class II
expression has been shown to have an effect on the net charge and
hydropathic index of the TCR CDR3 region within this
subset.25
Theoretically, two mechanisms of T-cell reconstitution are expected
after allo-BMT: First, peripheral expansion of graft-derived mature
(memory) donor T cells provides the first wave of T cells after
allo-BMT.26-30 These cells can be maintained in the
periphery for over 10 to 20 years.31,32 The second
mechanism involves thymic and/or extra-thymic selection processes and
expansion of positively selected donor precursor T cells. The latter
process of T-cell selection probably accounts for the more durable
reconstitution of the T-cell immune repertoire.33-35 The
MHC class II-mediated selection may present some special problems in
an MHC class II-deficient environment with respect to the generation
of a fully competent T-cell immune repertoire. In particular, positive
and negative selection events after allo-BMT may be hampered due to the
lack of MHC class II expression in these BLS patients on the thymic epithelial cells of the cortex and medulla, which are not of
hematopoietic origin.19,36
Patients.
Between 1985 and 1995, six unrelated infants suffering from MHC class
II-deficiency were treated with an allo-BMT in the Department of
Pediatrics at the Leiden University Medical Center. Conditioning of the
patients was performed according to protocols of the European Society
for Immunodeficiencies (ESID) and the European Group for Bone Marrow
Transplantation (EBMT). Only two of the transplanted patients showed
successful engraftment with immunological recovery and are alive from
1.5 to 4 years after allo-BMT. These unrelated patients: Patients 1 (unique patient number [UPN] 235; OSE) and 2 (UPN 293; EBA) who
received transplants in 1993 and 1995, respectively, with a full BM
graft from their healthy HLA-identical sibling donors: Donors 1 [D(UPN235); MSE] and 2 [D(UPN293); CBA] were analyzed in this
study. The characteristics of these patients, the transplant-related
variables, and the allo-BMT outcome are presented in
Table 1. The BM graft consisted of 4.6 × 108 nucleated cells/kg BW in the case of
patient 1, and 2.5 × 108 nucleated cells/kg body
weight (BW) in the case of patient 2. Acute graft-versus-host disease
(GVHD) grade I with involvement of the skin was observed in patient 1 and was lacking in patient 2. Limited chronic GVHD was observed in
patient 1 after allo-BMT and was absent in patient 2. Both children
received intravenous Igs immediately after allo-BMT, ie, patient 2 for
1.5 months, after which supplementation was discontinued. At the time
of this study, patient 1 was still on Ig supplementation. The chimerism patterns after allo-BMT were determined via a polymerase chain reaction
(PCR)-based CA repeat analysis in
fluorescence-activated cell (FACS)-sorted cell populations, adapted
from Van Leeuwen et al.38 For the purpose of this study,
both BM donors and recipients received a TT (booster) vaccination at
approximately 1 year after allo-BMT, and blood was drawn 4 weeks after
the vaccination. The use of this human material has been approved by
the Committee on Medical Ethics of the Leiden University Medical Center
(Protocol: P254/96).
Processing of PBMCs.
Approximately 20 mL of heparinized blood was drawn from the BLS
patients before allo-BMT and from the donors and allo-BMT recipients 4 weeks after TT booster vaccination. Peripheral blood mononuclear cells
(PBMCs) were separated over a Ficoll-Isopaque gradient (LUMC Hospital
Pharmacy, Leiden, The Netherlands). Immunophenotypical analysis of PBMCs was performed at regular intervals after allo-BMT as
described previously.38 Normalization of a lymphocyte
subset was defined as reaching the fifth percentile of age-matched
reference values.39,40 Approximately 2 × 106 PBMCs of both donor and recipient was used for
FACS-sorting to separate the CD4+ and CD8+
T-cell subsets using fluorescein-conjugated anti-CD4 and
R-phycoerythrin-conjugated anti-CD8 monoclonal antibodies (MoAbs)
(DAKO, Dakopatts, Glostrup, Denmark) and a FACScan (Becton Dickinson,
Mountain View, CA). The recovered CD4+ and CD8+
T cells were 98% to 99% pure. CD4+ and CD8+
single positive T-cell lines were generated by culturing Antigen-specific T-cell lines/clones.
For the analysis of the antigen-specific repertoire, TT-specific T-cell
lines were generated as described previously.42 Briefly, 3 to 6 × 106 PBMCs were grown in culture medium in the
presence of 1.9 limes flocculationis (lf)/mL TT
(RIVM, Bilthoven, The Netherlands). After two rounds of stimulation and
subsequent TT-specificity testing in a 3H-thymidine
incorporation assay, T-cell clones were generated from these lines by
limiting dilution.43 The T-cell lines and clones that,
after repeated testing, had a stimulation index (SI) > 3, with more
than 500 counts per minute, were considered to be specific for TT (SI = 3H incorporation of T cells with APC + TT/3H
incorporation of T cells with APC only) and used for further molecular
analysis. MHC class II-positive B-cell lines derived from the healthy
HLA-identical siblings were used as antigen-presenting cells (APC) in
these experiments.
RNA extraction, cDNA synthesis, PCR amplification.
Total RNA was extracted from TT-specific T-cell clones, and
CD4+DR Spectratyping.
The distribution of the TCR CDR3 sizes was analyzed by
PCR.41 For these purposes, a PCR reaction was performed
with 5' TCRBV family specific primers (TCRBV 1-23) and a 3'
TCRBC internal primer labeled with [ PCR fragment purification, DNA sequencing.
The PCR fragments were purified by electrophoresis in a 1%
low-melting-point agarose gel. The desired fragment was isolated and
purified using Wizard Columns (Promega) and used for direct sequencing47 based on the dideoxy-nucleotide chain
termination method.48 The sequencing reactions were done
with the T7 sequencing Kit (Pharmacia LKB, Uppsala, Sweden) using 5 to
10 pmol of TCRAC or TCRBC internal primer (listed below), approximately
0.25 pmol of PCR fragment, and [ Oligonucleotide sequencing/spectratyping primers.
The constant region sequencing and spectratyping primers used were:
TCRAC internal 5' GGT ACA CGG CAG GGT CAG GGT TC 3'; and TCRBC internal 5' TGT GGG AGA TCT CTG CTT CTG 3'.
Standard immunophenotypical analysis and monitoring of T-cell
proliferative responses before and at 1 year after allo-BMT.
Standard immunophenotypical analysis of PBMCs
(Table 2) showed that both
BLS patients had a normal percentage of CD3+ T cells before
allo-BMT. In patient 1, a decreased percentage of CD4+ T
cells and an increase of CD8+ T cells was observed, whereas
in patient 2 normal percentages of both T cell subsets were present. At
1 year after allo-BMT the percentage of CD3+ T cells was
low to normal, the percentage of CD4+ T cells was low in
both patients, and the percentage of CD8+ T cells was
either normal (patient 1) or increased (patient 2) when compared with
age-matched healthy controls.39,40 Analysis of CD45RA/RO
expression50 showed that in patient 1 before allo-BMT the
majority of the CD4+ T cells was of the CD45RO+
memory-phenotype, whereas the majority of T cells in patient 2 and the
CD8+ T cells of patient 1 was of the CD45RA+
naive-phenotype (Table 3). After allo-BMT,
a substantial population of the CD4+ (27% to 43%) and of
the CD8+ T cells (54% to 85%) was of the
CD45RA+ naive-phenotype in both allo-BMT recipients (Table
3).
MHC expression patterns and chimerism analysis.
FACS analysis of the PHA/rIL-2-activated CD4+ and
CD8+ T-cell subsets in both BLS patients before BMT showed
an absence of MHC class II (HLA-DR) expression and dull expression of
MHC class I when compared with the donors as shown in
Fig 1. A similar analysis after allo-BMT
revealed a deficiency in the induction of MHC class II in both
recipients, in particular in the CD8+ T-cell subset (Fig
1). Both BM donors showed approximately 70% MHC class II-positive
cells in both T-cell subsets. In patient 1, after allo-BMT, expression
of MHC class II was observed in 24% of the CD4+ T cells,
whereas 5% of the CD8+ T-cell subset was MHC class
II-positive. Similar observations were made in patient 2, after
allo-BMT, with 46% and 15% MHC class II-positive T cells in
CD4+ and CD8+ T-cell subsets, respectively.
However, in contrast to MHC class II, other activation markers, such as
CD25 and CD45RO, were found to be expressed at normal levels in both
allo-BMT recipients (results not shown). Because recipient-derived T
cells lack the capacity to express MHC class II upon activation, a
chimerism analysis was performed to determine the origin of these MHC
class II-negative T-cell fractions. These analyses revealed a mixed
recipient-donor T-cell chimerism in patient 1 (Table 4). Approximately 40% of the
CD4+ T cells and less than 10% of the CD8+ T
cells were of donor origin (Table 4), and all other hematopoietic lineages showed a similar distribution (Table 1). Patient 2 displayed a
different phenotype, as the majority of the hematopoietic cell lineages
were found to be of donor origin (Tables 1 and 4). Investigation of MHC
class I expression in these T-cell subsets (Fig 1) supported the
observation based on chimerism analysis as described above. Together,
these data indicate that after allo-BMT, in particular, donor-derived
CD8+ T cells are hampered in the induction of MHC class II
expression after T-cell activation.
TCR CDR3 size distribution patterns.
To study the overall TCR reconstitution in the periphery in more
detail, we analyzed the TCR
Generation of TT-specific T-cell lines and clones.
After stimulation of PBMC derived from patients 1 and 2 after allo-BMT
with TT, the responding TT-specific T cells showed specificity for TT
in a 3H-thymidine incorporation assay with SI ranging from
7 to 30 (Table 5). This was found to be in
a similar range when compared with their donors. Mitogenic stimulation
of these donor or recipient T-cell lines with PHA showed a similar
proliferative capacity (SI 48 to 84) as shown in Table 5. Subsequently,
TT-specific T-cell clones were generated from these T-cell lines by
limiting dilution. The percentage of TT-specific T-cell clones derived from these lines varied from approximately 10% in donor-recipient couple 1 to approximately 45% in donor-recipient couple 2 (Table 5,
right panel), all of which exhibited a similar proliferative capacity
(results not shown). Furthermore, all generated TT-specific T-cell
clones demonstrated a donor phenotype since they expressed MHC class II
upon activation.
Molecular characterization of TT-specific T-cell clones.
When comparing TCRBV sequences of TT-specific T-cell clones derived
from the two BM donors, a marked overrepresentation of TCRBV5
containing TCRs was observed. This preferential utilization of TCRBV5
was not found after transplantation in the allo-BMT recipients
(Table 6). Furthermore, no apparent sharing
of TCRBV, TCRBJ, and amino acid composition of the TCR CDR3 region was
observed between donor- and recipient-derived sequences (Table 6). When analyzing the DNA sequences of the TT-specific T-cell clones in more
detail, no striking differences were observed in the length of the TCR
CDR3 region (Table 6) or in the amount of nongermline modification at
the sites of recombination between donor and recipient (results not
shown). T-cell clones with an identical
The aim of this study was to investigate the T-cell immune
reconstitution after allo-BMT in an environment lacking endogenous MHC
class II. For these purposes, we have analyzed the functional and
phenotypical properties of the overall peripheral TCR repertoires, as
well as of the TT-specific TCR repertoires in two MHC class II-deficiency patients.
We thank Drs S.J.P Gobin, R.R.P. de Vries, I.I.N. Doxiadis, G.M.Th.
Schreuder (Leiden University Medical Center), and J.T. Kurnick
(Massachusetts General Hospital and Harvard Medical School, Boston, MA)
for critically reading the manuscript, L. Wilson (Leiden University
Medical Center) for assisting with FACS analysis, and A. van de Marel
and M. van der Keur (Leiden University Medical Center) for FACS-sorting
and analysis.
Submitted November 23, 1998; accepted March 7, 1999.
Supported in part by the J.A. Cohen Institute for Radiopathology and
Radiation Protection (IRS).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Peter J. van den Elsen, PhD, Department of
Immunohematology and Blood Bank, Leiden University Medical Center, Bldg
1, E3-Q, Albinusdreef 2, PO Box 9600, 2300 RC Leiden, The Netherlands;
e-mail: pvdelsen{at}euronet.nl.
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TOP
ABSTRACT
INTRODUCTION
2 × 105 to 5 × 105 sorted cells of each
subset in RPMI 1640 medium containing 5% human AB serum supplemented
with 2 mmol/L L-glutamine, 100 IU/mL penicillin, 100 µg/mL
streptomycin, 20 IU/mL recombinant interleukin-2 (rIL-2), and 1 µg/mL
phytohemagglutinin (PHA) for 14 days. The TCR CDR3 size
distributions
and
-DR+,high T-cell fractions were investigated after a second
FACS-sort using fluorescein-conjugated anti-HLA-DR MoAb (Becton
Dickinson) in combination with R-phycoerythrin-conjugated anti-CD4 or
anti-CD8 MoAbs (Dakopatts). To avoid contamination with
HLA-DR+,dull T cells, only truly HLA-DR
-32P]ATP as
described previously.
-33P]dATP (0.5 µCi)
(Dupont New England Nuclear Research Products, Boston, MA). DNA
sequences were compared with TCR sequences contained in the GenBank
using the PCGENE Computer Software Program (Release 6.85;
Intelligenetics Inc, Palo Alto, CA). The hydropathic index for amino
acids within the CDR3 component of each different TCRBV rearrangement
of the TT-specific T-cell clones was calculated using the SOAP program
included in the PCGENE computer software program. The resultant figure
for the amino acid sequence is given under the name of Grand Average of
Hydropathicity (GRAVY). The CDR3 region was defined for the purpose of
this analysis as being the number of amino acids between five amino
acids before the conserved cysteine at position 92 in the TCRBV
element
-HLA-DR); GAM-FITC was used as a negative control.
-chain CDR3 size profiles. Both BLS
patients before and after allo-BMT showed similar TCR CDR3 size
distribution patterns, as demonstrated by the results of patient 1 (Fig 
) T-cell
fractions. (C) Donor 1. From left to right, TCRBV families 1-23 are
depicted in CD4+ and CD8+ T-cell subsets.
