6.11
CLINICAL SCIENCE SESSION 10/17/2001
CD4+CD25+ REGULATORY CELLS IN ACQUIRED TRANSPLANTATION
TOLERANCE
EH Field, D Matesic, S Rigby, T Fehr and T
Rouse. Department of Veterans Affairs Medical Center, and Department of
Internal Medicine, University of Iowa College of Medicine
CD4+
cells that regulate the development of transplantation tolerance have been
described in several models of acquired tolerance. However, relatively
little is know about the actual phenotype of the regulatory cell, other
than they appear to be CD4+, or about the mechanism by which the regulatory
cells function to maintain the tolerant state to foreign transplantation
antigens. We have studied the phenotype and function of regulatory CD4+
cells in acquired tolerance to disparate class I and class II MHC in a
model of neonatal tolerance. To generate tolerant mice, we i.p. injected
neonatal BALB/c mice (< 24 hr old) with live semi-allogeneic CAF1 (BALB/c
X A/J) splenocytes. Mice were grafted with fully allogeneic A/J or third-party
B6 skin when they reached 4-6 weeks of age. Up to 70% of the neonatally-injected
mice maintained the A/J skin grafts for > 60 days, but rejected third-party
B6 skin. Absence of rejection was confirmed by histology. Adoptive transfer
experiments demonstrated that neonatal tolerant mice contained CD4+cells
that regulate the development of CD8+ T-cell alloreactivity. Co-injection
of unfractionated (but not CD4-depleted) spleen cells from tolerant mice
along with syngeneic CD8+ cells from naïve immunocompetent mice into
SCID mice that have A/J skin grafts in place, impaired the ability of the
normal CD8+ T-cells to reject the A/J skin grafts. Regulatory CD4+ cells
from tolerant mice were further phenotyped as CD4+CD25+ cells which expressed
CTLA4. We used BrdU incorporation, standard CTL assays, short-term IFNgamma
ELISPOT, and intracellular FACS analysis to study the effect of regulatory
CD4+ cells on the in vitro development of CD8+ T-cell alloreactivity. Neonatal
tolerant mice contained CD4+CD25+ cells that suppressed the development
of anti-donor CD8+ T-cell responses in vitro, including proliferation,
and generation of anti-donor CTL and TC1 responses. However, CD8+ T-cells
from tolerant mice partially recovered their capacity to generate anti-donor
effector cells (CTL and TC1) in vitro when the CD4+ regulatory cells were
removed prior to culture, as long as the cultures were also supplemented
with IL-2. This suggested that regulatory CD4+ cells functioned to maintain
alloreactive CD8+ effector T-cells in a state of functional anergy.We
conclude that regulatory CD4+CD25+ cells initiate and/or maintain tolerance
by preventing the development of CD8+ T-cell alloreactivity.