Research Report 2000 - MDC

Molecular
Immunology and
Gene Therapy
Thomas Blankenstein
Interleukin 4-deficient mice
reconstituted with wild-type
bone marrow fail to produce
normal immunoglobulin E levels
The ability to reconstitute interleukin
(IL)-4 -/- mice with bone marrow from
IL-4 +/+ mice was investigated. The
absence of the IL-4 -/- gene in donor or
recipient cells did not impair the
reconstitution. All immunoglobulin
(Ig) subsets occurred at normal serum
levels, except for IgE and to some
extent IgG1. IgE production did not
recover in the reconstituted mice over
a long period. However, these mice
were competent for IgE production,
because a single intrasplenic injection
of IL-4 restored IgE levels, which
then remained constant. Wild-type
mice reconstituted with wild-type
bone marrow maintained IgE serum
levels comparable with untreated
animals. In wild-type mice
reconstituted with IL-4 -/- bone marrow,
IgE levels decreased gradually and
disappeared after 12 weeks. We have
made three unrelated, but nonetheless
important, conclusions:
(a) (immunoregulation) the tightly
regulated IL-4 gene is expressed
continuously in low amounts (and
with apparent absence of antigen
stimulation) to maintain the normal
threshold of IgE; (b) (ontogeny of the
immune system) an early unidentified
source of IL-4 is postulated which is
lost in adult mice; and (c) (bone
marrow transfer/gene therapy) under
certain circumstance, the genotype of
the recipient influences the
reconstitution.
B cells inhibit induction of
T cell-dependent tumor
immunity
Cytotoxic T lymphocyte (CTL)
mediated tumor immunity against
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major histocompatibility antigen
(MHC) class I + but class II - tumors
often requires help from CD4 + T cells.
These CD4 + T cells are activated by
MHC class II + cells that present tumor
derived antigens. Considering that
different antigen presenting cells
(APC), such as B cells, macrophages
and dendritic cells, compete for
antigen and influence the outcome of
an immune response, we have
examined tumor immunity in B cell
deficient mice and showed that the
low immunogenicity of tumors is
caused by B cells whose presence in
the priming phase results in disabled
CD4 + T cell help for CTL-mediated
tumor immunity. Instead, in the
presence of B cells, a non-protective
humoral immune response is induced.
Our results may explain the enigmatic
observation that tumor-reactive
antibodies occur frequently in cancer
patients.
Direct and indirect T cell
priming by dendritic cell
vaccines
The mechanisms by which dendritic
cell (DC) vaccines prime host T cells
in vivo has been examined. Mice were
immunized with syngeneic bone
marrow-derived DC and βgalactosidase
(β-gal) was used as a
surrogate antigen. DC, either pulsed
with peptide, loaded with β-gal
antigen or gene-modified, induced βgal-specific
CTL and moderate
rejection of an in vivo challenge with
β-gal expressing tumors. In addition,
β-gal-specific CTL lysed the
syngeneic DC that were used as
vaccines. Using SCID mice
reconstituted with F1 lymphocytes,
direct priming by gene-modified DC
vaccines was demonstrated by the
presence of β-gal-specific CTL of the
haplotype exclusively expressed by
DC, while indirect priming by host
APC was shown by the detection of
CTL of the haplotype exclusively
present on host APC and absent on
DC vaccines. DC in vitro by
lymphokine-activated killer cells, DC
vaccines appear to interact with host
natural killer cells as well as with
antigen-specific T cells. These effector
cells, in turn, may lyse DC vaccines,
thereby, leading to the release of
antigens that can be taken up by host
APC.
TH1 associated and cytotoxic T
lymphocyte-mediated tumor
immunity is impaired in IL-4
deficient mice
Cellular immune responses are
induced by CD4 + T helper 1 (Th1)
cells secreting interleukin (IL)-2 and
interferon (IFN)-γ. Tumor immunity is
often mediated by CTLs whose
activation is supported by Th1
cytokines. Since IL-4 directs Th2
development, and has been shown to
inhibit Th1-dominated responses, we
have assumed that IL-4-deficient
(IL-4 -/- ) mice would develop vigorous
CTL-mediated tumor immunity
compared with IL-4-competent
(IL-4 +/+ ) mice. Surprisingly, IL-4 -/mice
exhibited a severely impaired
ability to develop tumor immunity.
The lack of tumor immunity in IL-4 -/mice
was associated with reduced
IFN-γ production, diminished levels
of tumor-reactive serum IgG2a, and
undetectable CTL activity, indicating
a defective Th1 response in the
absence of endogenous IL-4. Anti-IL-
4 monoclonal antibody blocked tumor
immunity in IL-4 +/+ mice when
administered at the time of
immunization but not at the time of
challenge. Additionally, tumor
immunity could be induced in IL-4 -/mice,
if IL-4 was provided by genemodified
cells together with
immunizing tumor cells. These results
demonstrate that tumor immunity
requires IL-4 in the priming phase for
the generation of effector cells rather
than for their maintenance. Together,
our results demonstrate a novel, and
previously unanticipated, role of IL-4
in the generation of Th1-associated,
CTL-mediated tumor immunity.
Retroviral gene transfer
We have constructed retroviral vectors
carrying marker genes such as
β-galactosidase and green fluorescent
protein and have optimized retroviral
gene transfer into different cell types.
We have analyzed the retrovirus
receptor expression on different
human tissues and cell lines and
shown that the amount of receptor
expression does not correlate with the
transduction efficiency of three
retrovirus vector pseudotypes
(A-MuLV, GALV, 10A1) using these
receptors for cell entry. We have
generated retrovirus vectors carrying
‘suicide’ genes encoding cytosine
deaminase and HSV thymidine kinase
and transferred these genes into