T-cell costimulation in graftversus- host disease and graft-versus-leukemia effect - Immune Biology of Allogeneic Hematopoietic Stem Cell Transplantation - page 208

In addition to naturally derived Tregs (nTregs), CD4 + CD25 + FOXP3 + cells can
also be generated or differentiated from naïve CD4 + T cells in the periph-
ery and are termed induced Tregs (iTregs). Several groups including ours
showed that CD28 but not ICOS signaling is required for the differentia-
tion of iTregs from naïve CD4 + CD25 − T cells. The requirement of CD28 for
Treg differentiation was mediated by IL-2 but not other γ-chain cytokines,
because neutralization of IL-2 with its specific mAb blocked Treg differenti-
ation from WT T cells and addition of IL-2 restored Treg differentiation from
CD28 −/− T cells [45] . However, using a series of transgenic mice on a CD28-
deficient background that bears either WT or mutated CD28 in its cytosolic
tail incapable of binding to Lck, PI3-kinase or Itk, we found that strong
CD28 costimulation suppresses iTreg induction through Lck signaling inde-
pendent of IL-2 production. Furthermore, suppression of iTreg generation
through CD28-mediated Lck signaling contributes to T-cell pathogenicity
in the induction of GVHD [149] .

As observed previously, Tregs constitutively express high levels of CTLA4,
suggesting that CTLA4 may be involved in their suppressive function.
Early evidence that supports the role of CTLA4 in regulating Tregs came
from the observation that Tregs were required to control lymphoprolif-
erative disease caused by CTLA4 KO T cells [150] . More recent studies
confirmed a dual role for CTLA4 on Teffs and Tregs and further showed
that CTLA4 on Tregs was critical to prevent activation of autoreactive
Teffs in lymphoid tissues, but CTLA4 on Teffs prevented infiltration and
destruction of peripheral tissues [151,152] . Importantly, Wing et al. [153]
showed that a fatal lymphoproliferative disease still develops in CTLA4
conditional-KO mice, with a deficiency of CTLA4 specifically in FOXP3-
expressing Tregs, despite slower kinetics, clearly demonstrating a dual
role for CTLA4 on Teffs and Tregs in maintaining T-cell homeostasis and
tolerance. However, CTLA4 is dispensable for the development, sur-
vival, and homeostasis of Tregs, as normal numbers and percentages of
CD4 + CD25 + FOXP3 + Tregs are found in the absence of CTLA4 [153] . The
mechanism by which Tregs induce immune suppression through CTLA4
is through ligation of B7 on APCs by CTLA4 on Tregs, inducing indole-
amine 2,3-dioxygenase, an enzyme that catabolizes tryptophan in the
extracellular milieu, which inhibits T-cell proliferation [154,155] . Recent
data suggest that CTLA4 expressed by Tregs may inhibit APCs by strip-
ping off B7 molecules, thereby inhibiting CD28-mediated costimulation
to Teffs [153] . Importantly, the downmodulation of B7 expression required
direct contact between Tregs and APCs and was dependent on the pres-
ence of CTLA4 on Tregs [153,156] . The role of CTLA4 in GVHD through a
direct effect on Tregs has not been formally tested, but CTLA4 should not
be blocked in GVHD prevention, given its dominant negative role in regu-
lating Teffs but requirement for Treg suppressive function.

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Using WT or CD28, ICOS, or CD28/ICOS-double KO mice, we found that
CD28 plays a major, whereas ICOS plays a minor, role in the development
of and homeostasis of nTregs [45] . We and others also found that ICOS is
dispensable for the generation and function of induced Tregs in vitro and
in vivo [45,157] . However, in a mouse model of type 1 diabetes (T1D), block-
ade or genetic deficiency of ICOS in NOD mice exacerbates T1D by abro-
gating Treg-mediated function and T1D protection [158,159] . Furthermore,