results are explained by a rheostat mechanism
whereby CD45 differentially regulates the nega-
tively acting pTyr-505 and positively acting
pTyr-394 p56
lck
tyrosine kinase phosphorylation
sites. We propose that high wild-type CD45
expression is necessary to dephosphorylate
p56
lck
pTyr-394, suppressing CD4 T
+
cell line-
age commitment and hyperactivity.
INTRODUCTION
The transmembrane and heavily glycosylated CD45
tyrosine phosphatase (PTPase) has attracted attention
because of its high expression on the surface of T and
sponses from high in the thymus to much lower in the
periphery (Grossman and Singer, 1996; Sebzda et al.,
1996). The threshold of TCR engagement required for
activation is reduced even further in Ptprc
+/
(encoding
CD45) mice thymocytes in which CD45 PTPase activity
is lower and positive selection is amplified (Wallace
et al., 1997). The varying concentration of CD45 in specific
subcellular localizations has also received attention in the
context of signaling studies on the immune synapse and
on microclusters (Campi et al., 2005; Douglass and
Vale, 2005; Varma et al., 2006; Yokosuka et al., 2005).
Such findings have stimulated the focus of our present
study, which is on the differential expression of CD45
and the way in which the consequent titration of its phos-
phatase activity is critical in dictating TCR signalingImmunity
Article
The Differential Regulatio
Phosphorylation Sites by
for T Cell Receptor Signa
Louise McNeill,
1,3
Robert J. Salmond,
1,3
Joanne C. Coo
Marta Roche-Molina,
1
Panna Tandon,
1
Nick Holmes,
2
a
1
Laboratory of Lymphocyte Signalling and Development, The Ba
2
Department of Pathology, University of Cambridge, Tennis Cou
3
These authors contributed equally to this work.
*Correspondence: denis.alexander@bbsrc.ac.uk
DOI 10.1016/j.immuni.2007.07.015
SUMMARY
The molecular mechanisms whereby the CD45
tyrosine phosphatase (PTPase) regulates T
cell receptor (TCR) signaling responses remain
to be elucidated. To investigate this question,
we have reconstituted CD45 (encoded by
Ptprc)-deficient mice, which display severe
defects in thymic development, with five differ-
ent expression levels of transgenic CD45RO,
or with mutant PTPase null or PTPase-low
CD45R0. Whereas CD45 PTPase activity was
absolutely required for the reconstitution of
thymic development, only 3% of wild-type
CD45 activity restored T cell numbers and nor-
mal cytotoxic T cell responses. Lowering the
CD45 expression increased CD4 lineage com-
mitment. Peripheral T cells with very low activity
of CD45 phosphatase displayed reduced TCR
signaling, whereas intermediate activity caused
hyperactivation of CD4
+
and CD8
+
T cells. TheseB lymphocytes (Alexander, 2000; Hermiston et al., 2003;
Holmes, 2006). Why does an enzyme need to be so abun-
dant at the T cell surface? No physiologically relevant
exogenous CD45 ligands have yet been identified,
although various lectins have been suggested (van Vliet
Imn of Lck Kinase
CD45 Is Critical
ling Responses
per,
1
Ce´ line K. Carret,
1
Robin L. Cassady-Cain,
1
nd Denis R. Alexander
1,
*
braham Institute, Babraham, Cambridge CB2 4AT, UK
rt Road, Cambridge CB2 1QP, UK
et al., 2006). Considerable attention has been given to
the alternative splicing of CD45 in T cells, generating up
to eight different CD45 isoforms, differing only in their
ectodomains (Beverley et al., 1992). These isoforms
appear to differentially associate in cis with the CD4 and
CD8 coreceptors, thereby providing a feasible mecha-
nism whereby the CD45 phosphatase can act on CD4
and CD8-associated p56
lck
tyrosine kinase, which is crit-
ical for the initiation of T cell receptor (TCR)-mediated sig-
nals (Dornan et al., 2002; Leitenberg et al., 1996). Further-
more, CD45 isoforms can differentially homodimerize,
particularly at higher expression, possibly leading to inhi-
bition of the phosphatase activity, a finding with relevance
to autoimmunity (Majeti et al., 1998, 2000; Xu and Weiss,
2002). A further possible isoform-dependent mechanism
involves the differential targeting of CD45 to p56
lck
in lipid
rafts (Irles et al., 2003). Several reports have also high-
lighted the importance of CD45 expression in its differen-
tial regulation of TCR signaling amplitude. For example, it
is well established that CD45 expression increases during
T cell development, becoming 2-fold higher in the periph-
ery than in the thymus (McNeill et al., 2004). This corre-
lates with a change in the sensitivity of TCR signaling re-responses.
The most important substrate for CD45 in T cells is the
p56
lck
tyrosine kinase (Ostergaard et al., 1989), p59
fyn
also being a substrate (Mustelin et al., 1992). CD45 is
the only known phosphatase that dephosphorylates the
munity 27, 425–437, September 2007 ª2007 Elsevier Inc. 425

Figure 1. Effects of Altered PTPase Activity on T Cell Differentiation in Mutant CD45RO-PTPase Mice
(A) Cell-surface expression of CD45 on thymocytes from wild-type (gray histogram), Ptprc
/
(black histogram), and CD45RO-C817S, CD45RO-
V821S, and CD45RO9 as unfilled histograms from left to right, respectively.
(B) Thymic CD45 expression (diamond) and membrane PTPase activity (bar) presented as average percentage of wild-type (mean ± SEM, minimum
Immunity
CD45 Regulation of TCR Responsesof 5 mice for each group).
(C) Flow cytometry of DN thymocytes (gated CD4

CD8

cells) showing CD44 and CD25 expression. Numbers in quadrants indicate the frequency of
DN2 (CD44
+
CD25
+
) and DN3 (CD44

CD25
+
) thymocytes.
(D) Flow cytometry analysis of CD4 and CD8 expression on thymocytes with CD4
+
CD8
+
, CD4
+
SP, and CD8
+
SP thymocyte populations highlighted by
gating. Numbers indicate the frequency of cells in each gated population.
426 Immunity 27, 425–437, September 2007 ª2007 Elsevier Inc.