Download Cellular and Gene Therapy for Major Histocompatibility Complex

Cellular and Gene Therapy for Major
Histocompatibility Complex Class II Deficiency
Franck Matheux1,2 and Jean Villard1
1
Immunology and Transplant Unit, Division of Immunology and Allergology, Geneva University Hospital, 1211 Geneva 4; and 2Department of Genetics
and Microbiology, Geneva Medical School, 1211 Geneva, Switzerland
P
rimary immunodeficiency diseases represent a heterogeneous group of inborn errors of the immune system affecting children in their first years of life. All of these syndromes
are characterized by abnormalities in the development, maturation, or function of cells of the immune system. Although the
phenotypes of these hereditary immunodeficiency diseases
(typically characterized by an extreme susceptibility to infections) are fairly uniform, the genetic defects that are responsible are very heterogeneous. One example of primary immunodeficiency is called severe combined immunodeficiency
(SCID). Blood analyses of SCID children are characterized by
absence of T cells, B cells, and sometimes natural killer (NK)
cells. Several genetic defects have been demonstrated to lead
to the same phenotype. SCID can result from mutations in the
adenosine deaminase gene, which encodes for a protein
implicated in DNA synthesis and cell survival. SCID can result
in defects in the Janus tyrosine kinase (Jak) 3 or in genes
involved in the rearrangement of T and B cell antigen-specific
receptors (the RAG 1 and RAG 2 genes). SCID can also result
from a defect in the gene encoding a chain of the common
receptor of several cytokines [the common J-chain (Jc) gene].
This defect is called SCID-X1 (5).
Other well-known primary immunodeficiencies are characterized by a defect in antibody production. These include the
X-linked agammaglobulinemia, in which mutations in the
gene encoding Bruton tyrosine kinase lead to the disease. The
hyper-IgM syndrome is another example of an antibody production defect, wherein the B cells are not able to switch from
IgM to IgG, IgA, or IgE. The X-linked form of the disease
(called HyperIgM-1) is caused by an absence of the CD40
ligand (CD40L) protein. This protein is expressed at the cell
surface of the activated T cell, and the interaction between
CD40L and CD40, expressed at the cell surface of the B cell,
is crucial for the antibody switching in the germinal center of
lymph nodes (5). Major histocompatibility complex class II
(MHC II) deficiency is another primary immunodeficiency,
phenotypically homogenous but genetically heterogenous,
that will be described latter in more detail.
Most of these syndromes are severe, and bone marrow
transplantation (BMT) is currently the only therapy for curing
patients with primary human immunodeficiency. Unfortunately, most patients do not have histocompatible donors and
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10.1152/nips.01462.2003
receive alternate therapy with nonhistocompatible BMT,
which has a high rate of severe complications (5). Therefore,
the optimization of other therapies is necessary for children
suffering from these rare genetic disorders.
Transplantation of autologous hematopoietic stem cells
genetically modified to express the defective gene (stem cell
and gene therapy) is an alternative approach that may prove to
be superior to histocompatible BMT. In 2000, the first study of
gene therapy for children suffering from SCID-X1, which
accounts for more than half of SCID cases, was published. As
mentioned above, the SCID-X gene is caused by mutations of
the gene encoding a common receptor of several cytokines
(the Jc gene). The disease is very severe and is characterized by
the absence of both T and NK lymphocyte subsets from the
periphery. SCID-X1 represents the most suitable situation for
gene therapy because in one study (1) the correction of the
genetic defect relieved the block in early lymphoid development and provided a growth advantage to corrected T lymphocytes. In this study, the correction of hematopoietic cells
with a wild-type Jc gene led to spectacular positive results. The
T cells appear in the peripheral blood, and the immune functions were restored in most of these treated SCID-X1 patients.
Unfortunately, two of them developed leukemia several
months after the treatment. Insertion of the transgene in the
vicinity of the LM-only (LMO-2) gene has been detected. The
function of the LMO-2 protein is not clearly defined, but this
protein has been previously implicated in T cell leukemia (6).
These results provide the proof of concept that cellular and
gene therapy is a valid alternative to treat monogenic congenital disease, particularly primary immunodeficiency. However,
the two cases of leukemia clearly show that not all of the
processes are completely controlled. Monitoring of the insertion site is crucial, as is the acquisition of more experience in
animal models of human genetic diseases without an evident
selective growth advantage for transduced cells. Our group
has worked for many years on another primary immunodeficiency, MHC II deficiency, which is also a good candidate for
gene therapy protocol, as discussed below.
MHC II deficiency, a disease of gene regulation
MHC II antigens are cell surface molecules that play a piv0886-1714/04 5.00 © 2004 Int. Union Physiol. Sci./Am. Physiol. Soc.
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Major histocompatibility complex (MHC) class II deficiency is a primary immunodeficiency. Lentiviral vectors are used for gene therapy in a mouse model of this disease. In addition, by a direct
genetic correction approach, a diagnostic test to determine which of the four MHC II genes is
defective in new MHC II-deficiency patients has been optimized.
FIGURE 1. Elucidation of the molecular defects responsible for major histocompatability complex class II (MHC II) deficiency has led to the identification of four essential and specific transactivators of MHC II genes. A prototypical MHC II promoter is depicted with its conserved X, X2, and Y motifs.
The four transactivators affected in MHC II deficiency are highlighted. The
corresponding complementation groups, the percentage of patients in each
group, and the chromosomal location of the affected genes are indicated.
CIITA is a highly regulated non-DNA-binding coactivator that is responsible
for cell-type specificity and induction of MHC II expression. RFX5, RFXAP,
and RFXANK are the three subunits of the X box-binding regulatory factor X
(RFX) complex. All four genes are essential and highly specific for MHC II
expression. The other promoter-binding complexes (X2BP and NF-Y) are not
specific for MHC II expression and are not affected in MHC II deficiency.
genes (11). The genes that are affected in all four complementation groups have been identified (Fig. 1). The gene affected
in group A encodes the class II transactivator (CIITA). CIITA is
a differentially expressed coactivator protein that functions as
the master control factor determining the cell type specificity
and inducibility of MHC II expression (11, 14). The genes
affected in groups B, C, and D, encode RFXANK (8), RFX5
(13), and RFXAP (4, 16), respectively. These three proteins are
subunits of regulatory factor X (RFX), a ubiquitously expressed
protein complex that binds to the X box cis-acting sequence
present in MHC II promoters (Fig. 1). Defects in RFX account
for the majority (>80%) of all known patients. Isolation of the
genes that are defective in MHC II deficiency has provided us
with tools that are of obvious clinical relevance. The availability of the genes encoding CIITA, RFX5, RFXAP, and RFXANK
paves the way for the development of new approaches that
will improve the diagnosis and treatment of MHC II deficiency.
Rationale for envisaging gene therapy for MHC II
deficiency
The optimal symptomatic care currently available for MHC
II deficiency consists of the prophylactic use of antibiotics,
intravenous administration of immunoglobulins, and parenteral nutrition. Although these treatments can reduce the
frequency and severity of the clinical problems associated
with MHC II deficiency, they do not prevent progressive organ
dysfunction or death at a young age. As for other combined
immunodeficiency disorders, allogeneic BMT is therefore currently considered to be the treatment of choice. Unfortunately,
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otal role in the control of the immune response. They shape
the T cell repertoire by directing positive and negative selection in the thymus, and they initiate and regulate immune
responses by presenting antigenic peptides to the receptor of
CD4+ T helper lymphocytes. They also participate in the activation of the antigen-presenting cells on which they are
expressed. Considering these key functions, it is not surprising
that expression of MHC II molecules is tightly regulated and
largely restricted to professional antigen-presenting cells,
thymic epithelial cells, and cells exposed to a variety of stimuli, including immune regulators such as interferon-J. Precise
regulation of this expression pattern is crucial for the immune
system. This is emphasized by the fact that ectopic or abnormal MHC II expression has been implicated in the pathogenesis of autoimmune diseases, whereas the lack of MHC II
expression results in a severe immunodeficiency syndrome
called MHC II deficiency (11).
MHC II deficiency, also referred to as the bare lymphocyte
syndrome, is an autosomal recessive disorder. The disease
phenotype is due to the complete loss of MHC II expression in
all cell types. As expected from the central role of MHC II molecules, the most striking and constant immunologic characteristics of MHC II deficiency are the absence of cellular and
humoral immune responses to antigens. Patients are unable to
mount T cell-mediated immune responses to antigens that the
patients have been immunized with or sensitized to by infections. Humoral immunity is also severely impaired. Patients
are panhypogammaglobulinemic, almost agammaglobulinemic, or have a decrease in one or two immunoglobulin isotypes. Antibody responses to immunizations and infections by
microbial agents are generally absent or strongly reduced.
Although patients have normal numbers of circulating T and B
lymphocytes, CD4+ T cell counts are typically reduced and
CD8+ T cell counts are proportionally increased. This presumably reflects abnormal selection and maturation of CD4+ T
cells resulting from a lack of MHC II expression in the thymus
(7, 11, 17). The major clinical manifestations of MHC II deficiency are the same as those associated with other severe
immunodeficiency syndromes. These include septicemia and
recurrent infections of the gastrointestinal, pulmonary, and
upper respiratory or urinary tracts. The patients are prone to
bacterial, fungal, viral, and protozoan infections. These infections start during the first year of life, and subsequent evolution of the disease is characterized by an inexorable progression of the infectious complications until death ensues. Few
affected children reach puberty; the majority die between the
age of 6 mo and 5 yr (7, 11, 17).
Although the disease is a direct consequence of the lack of
MHC II expression, the primary genetic defects do not lie in
the MHC II genes themselves. Instead they reside in genes
encoding transacting regulatory factors required for the
expression of MHC II genes. These transacting factors activate
transcription of MHC II genes by interacting with a highly conserved DNA segment present in the promoters of all MHC II
genes (Fig. 1). Somatic cell fusion experiments performed with
cell lines derived from MHC II deficiency patients have
defined four different genetic complementation groups (A, B,
C, and D), reflecting the existence of four distinct regulatory
lentiviral vector expressing the mouse RFX5 gene. The lentiviral vector has been shown to be especially efficient to transduced hematopoietic stem cells (12).
After transduction, the cells are injected via the tail vein into
irradiated 6-wk-old RFX5/ mice. Following reintroduction
of the transduced cells into RFX5/ mice, repopulation of
these recipient mice by corrected bone marrow-derived stem
cells is analyzed by examining cell surface MHC II expression
in key cell types (B cells, dendritic cells, macrophages). The
restoration of a functional immune system in the recipient
mice is evaluated by analyzing their ability to mount B and T
cell-dependent immune responses (specific T cell activation,
production of antibodies, etc.) (Fig. 2).
Genetic correction of pluripotent stem cells is one of the
critical steps in gene therapy, because only these cells can initiate long-term reconstitution of the entire hematopoietic system. The phenotype of pluripotent stem cells is defined by several cell surface markers but mainly by the capacity of a few
of them to reconstitute all hematopoietic lineages for the long
term. Different options are possible to get pluripotent stem
cells from the bone marrow, which is obtained by flushing tibial and femoral bones of donor mice. Pretreatment of the
donor mice with 5’-fluorouracil is well known to enrich early
progenitors of the bone marrow. Another approach is to select
hematopoietic progenitors from the bone marrow. The purification of progenitors from the bone marrow of the RFX5/
mice can be obtained by cell sorting with a fluorescence-activated cell sorter (FACS) after the cells are labeled with the fluorescent markers of interest for hematopoietic stem cells.
The Sca1+ protein is one of these interesting cell surface
markers. It is expressed at the surface of bone marrow stem
cells but also at the cell surface of more differentiated cells
and in some mature bone marrow-derived cells. The selection
and isolation of Sca1+ cells from bone marrow match as
closely as possible the conditions currently used in the human
Cellular and gene therapy in a mouse model of MHC II
deficiency
In contrast to CIITA, which is tightly regulated, the RFX5
gene is expressed in every cell type that has been tested.
Potential problems related to ectopic transgene expression
have to be avoided in the context of gene therapy. It was therefore logical to choose the RFX5 knockout (RFX5/) mouse as
a model system to develop gene therapy for MHC II deficiency
(3). In MHC II deficiency, the key cellular compartments in
which MHC II expression should be restored by gene therapy
are bone marrow-derived antigen-presenting cells including
dendritic cells, B cells, and macrophages. To achieve this in
the RFX5/ mouse, we are using an ex vivo approach similar
to those that have been successful in other mouse immunodeficiency models, such as those for Jak3 deficiency, RAG-2
deficiency, Jc deficiency, or E-thalassemia. In the RFX5/
mouse model, bone marrow cells are isolated from a RFX5/
donor mouse. These cells are transduced ex vivo with the
supernatant of a retroviral vector that contains the mouse
wild-type RFX5 gene. For this model, the retroviral vector is a
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FIGURE 2. Experimental procedure for cellular and gene therapy in a mouse
model of MHC II deficiency. RFX5/ bone marrow stem cells are enriched
with 5’-fluorouracil (5-FU) or purified by cell sorting [fluorescence-activated
cell sorter (FACS) analysis]. The cells are then transduced with a lentiviral vector that contains the mouse RFX5 gene in the presence of cytokines such as
interleukin (IL)-1D, IL-3, stem cell factor (SCF), or thrombopoietin (TPO) for a
few hours. The transduced cells are then reinfused in lethally irradiated
RFX5/ mice to reconstitute all hematopoietic lineages. MHC II expression
and immune function and are analyzed after bone marrow reconstitution.
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the success rate of BMT for MHC II deficiency remains lower
than in other immunodeficiency syndromes. This does not
appear to be typical for MHC II-deficient patients. Instead, it is
likely due to other criteria such as diagnosis at a late age. The
two main obstacles are intractable persistent viral infections
and graft failure or rejection. The difficulty of finding allogeneic and human leukocyte antigen (HLA)-compatible
donors also represents a major problem (7). Given the poor
success rate of BMT for MHC II deficiency, the development
of an alternative curative therapy represents a major goal.
Now that four genes affected in MHC II deficiency have been
identified, treatment by cellular and gene therapy of all four
complementation groups can be envisaged.
Approaches relying on introduction of the wild-type CIITA,
RFX5, RFXANK, or RFXAP genes into hematopoietic stem
cells from patients in complementation group A, B, C, or D,
respectively, would represent a logical therapeutic strategy.
The validity and strength of this type of approach has recently
been emphasized by its resounding success in curing SCID-X1
infants carrying mutations in the Jc gene (1). In addition, many
features render MHC II deficiency an excellent candidate disease for the development of somatic gene therapy. First, thanks
to the isolation of all four affected genes, the molecular genetics of the disease is very well defined. Second, the genes
implicated in the majority of patients (RFXAP, RFXANK, and
RFX5) are constitutively expressed. Third, RFX5- and CIITAdeficient mice are available and both represent excellent animal models for the human disease (2, 3). They reproduce all
of the major features typical of MHC II deficiency, including a
severely disturbed pattern of MHC II expression and a profound deficiency in CD4+ T cell-dependent immune
responses. Fourth, pluripotent hematopoietic stem cells are
the relevant target cells that need to be corrected: the ability
of BMT to cure the disease has demonstrated that the correction of non-bone-marrow-derived cells, such as thymic epithelial cells, is not essential.
of the reporter molecule (GFP) by FACS in >50% of the transduced cells has been observed (Fig. 3A). Subsequently, the
lentiviral vectors encoding CIITA, RFXANK, RFX5, and RFXAP
have been used to correct primary cells derived from MHC IIdeficiency patients (Fig. 3B).
This approach permits the classification of several new
MHC II-deficiency patients. Representative data for a group D
patient is shown in Fig. 3. PBL from this patient were cultured
and transduced with lentiviral vectors containing the four
genes. After 7 days of culture, 48% of the cells were MHC II
positive in the RFXAP well only (Fig. 3B) (9). The direct genetic
correction is a novel diagnostic tool that could certainly be
extended to other primary immunodeficiencies or to other disorders characterized by a genetic heterogeneity.
Conclusions
Direct genetic correction for diagnosis and molecular
characterization of MHC II deficiency
However, the correction of a genetic defect can also be useful for other purposes than gene therapy. The hallmark of MHC
II deficiency is the absence of MHC II molecules on the surface of all cells, and the demonstration of this lack of expression currently remains the mainstay of diagnosis. However,
this approach does not identify the precise genetic defect that
is responsible for the absence of MHC II expression. Until
now, classification of the patients into the genetic complementation groups has been done by isolating stable cell lines
from the patients and performing tedious and time-consuming
cell fusion experiments. In addition, the availability of a simple and rapid approach for determining which regulatory
genes are affected in newly identified patients will be a crucial step for the success of cellular and gene therapy in this
disease. Now that the genes affected in all four complementation groups have been identified, a rapid and straightforward
approach relying on direct correction of the genetic defect has
been developed. We have set up a system in which cells
derived from newly identified MHC II-deficiency patients are
transduced with lentiviral vectors expressing the four regulatory genes (CIITA, RFXANK, RFX5, or RFXAP). This has been
done with readily accessible cell types, such as peripheral
blood lymphocytes (PBL). Complementation of the genetic
defect in the transduced cells can be scored very simply by
FACS analysis of cell surface MHC II expression a few days
after transduction of the cells. The procedure is thus designed
to provide a very reliable, rapid, and simple means of determining which of the four regulatory genes is defective. Transduction and gene delivery with lentiviral vectors encoding
CIITA, RFXANK, RFX5, and RFXAP were first optimized to
restore MHC II expression in cell lines established from
patients having defects in the corresponding genes (complementation groups, A, B, C, and D, respectively) (9, 10, 18).
Once the system had been optimized with cell lines, experiments were initiated with primary cell isolates (PBL). Since
MHC II deficiency patients are rare and not always readily
available, the transduction efficacy of primary cells isolated
from healthy individuals can be first evaluated with green fluorescent protein (GFP) lentiviral vectors. A strong expression
MHC II deficiency, like other primary immunodeficiencies,
FIGURE 3. Complementation of peripheral blood lymphocytes (PBL) from an
MHC II-deficiency patient in complementation group D. A: efficient transduction of PBL with lentiviral vectors. PBL from a normal donor or from a
patient were transduced with a control lentiviral vector encoding green fluorescenct protein (GFP). Expression of GFP was analyzed by FACS. Open profiles, untransduced cells; closed profiles, transduced cells. B: PBL are complemented by the RFXAP vector. PBL from the patient were transduced with
the four lentiviral vectors. The RFXAP vector restores cell surface MHC II
expression on 48% of the cells. Open profiles, untransduced cells from the
patient; closed profiles, transduced cells; shaded profile, PBL from a wildtype donor. HLA DR, human leukocyte antigen class II.
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setting, because no 5’-Fu can be used before bone marrow
harvesting and Sca1+ purification is a procedure partially
comparable with CD34+ human bone marrow purification. In
addition, the reconstitution of all bone marrow lineages (especially mature T lymphocytes) is faster with Sca1+ cells compared with the reconstitution with more highly purified stem
cells. Finally, highly purified transduced murine bone marrow
progenitors (defined by the surface markers Sca1+c-Kit+Lin
expressed by bone marrow stem cells ) are able to reconstitute
normal, multilineage specification (15) with very few cells.
This is certainly the best option for ex vivo gene therapy
approaches. The transduction of these cells with vectors that
contain the wild-type gene is the only way to obtain long-term
expression of the transgene and prolonged the correction of
the defect.
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is a good model for molecular medicine studies because the
disorder represents simple models that can be used in the
future for more complex multifactorial diseases such as
autoimmune diseases, allergy, and many other diseases.
Although disease manifestations cannot in all cases be
explained by single molecular events, their analysis shows
that a constant interplay between clinical and basic immunology can bring about fruitful results of mutual benefit.
Following the successful treatment of X-linked SCID, and
despite the dramatic complications for two patients, cellular
gene therapy remains a promising strategy, and encouraging
results have been demonstrated both in vitro and in vivo. Several obstacles remain to be overcome, such as the level of
expression of the transgene and the site of integration. Several
strategies are being applied to improve these problems.
The proof of concept that cellular and gene therapy can be
a valid alternative for untreatable human diseases has been
demonstrated. Animal models that closely mimic human diseases represent an invaluable tool and one of the key steps of
the approach. The principle of gene therapy is the correction
of a genetic defect. This approach can also be used as a very
useful tool for the diagnosis of heterogeneous human genetic
diseases.