Subsets of Nonclonal Neighboring CD4+T Cells
Specifically Regulate the Frequency of Individual
Antigen-Reactive T Cells
Nevil J. Singh,1,* Jennifer K. Bando,2and Ronald H. Schwartz1
1Laboratory of Cellular and Molecular Immunology, NIAID, NIH, Bethesda, MD 20892, USA
2Department of Microbiology and Immunology, University of California, San Francisco, CA 94143, USA
After an immune response, the expanded population
of antigen-specific CD4+T cells contract to steady
state levels. We have found that the contraction is
neither cell-autonomous nor mediated by competi-
tion for generic trophic factors, but regulated by rela-
tively rare subsets of neighboring CD4+T cells not
necessarily of a conventional regulatory T cell
lineage. These regulators, referred to as deletors,
specifically limit the frequency of particular antigen-
specific T cells even though they are not reactive to
deletor could outcompete the target for recognition
of a shared, nonstimulatory endogenous peptide-
MHC ligand. This mechanism was sufficient to
prevent even agonist-driven autoimmune disease in
a lymphopenic environment. Such a targeted regula-
tion of homeostasis within narrow colonies of T cells
with related TCR specificities for subthreshold
ligands might help to prevent the loss of unrelated
able diversity of the repertoire.
The number of T cells in the peripheral immune system is tightly
regulated in health and disease. In the steady state, homeostatic
processes maintain a stable population of helper T cells,
balancing thymicoutput with normalattrition (Freitas and Rocha,
2000). Infections trigger a dramatic expansion of otherwise rare
antigen-specific T cells, but this is transient and the population
density is restored soon after the pathogen is cleared. Further-
more, a separate set of processes ensure that T cells capable
of reacting to self-antigens are eliminated from the population
by clonal deletion (Gardner et al., 2008). These various elimina-
tion mechanisms must also be discriminating enough to ensure
that a diverse set of T cell receptors (TCRs) are still retained in
the peripheral repertoire in order to maintain defenses against
as wide a variety of future infections as possible. Given that
each T cell response yields a large frequency of expanded path-
ogen-specific T cells, if the subsequent contraction was regu-
lated by stochastic processes, it could also lead to a large loss
of unrelated ‘‘bystander’’ T cells and therefore a progressive
loss of repertoire diversity over multiple infections. The cellular
mechanisms that ensure such precise homeostatic control,
especially for CD4+T cells, are not clear.
In the last two decades, reductionist approaches to study
this complex problem have focused on understanding the regu-
lation of T cell survival. These studies have coalesced around
a conceptual framework based on competition for limiting
trophic resources, keeping T cell subsets within certain popula-
can allow the antigen-specific T cell numbers to exceed these
limits but the population returns to competing for the limiting
interactions after antigen clearance. The critical trophic factors
that anchor this process can be segregated into two categories:
public and cognate. The former are sensed by receptors not
related to the TCR and therefore do not respect the antigen
specifities of the T cells competing for them. These include cyto-
kines, such as interleukin-2 (IL-2), IL-7, and IL-15, and thymic
stromallymphopoietin (TSLP),aswellasnutrients, costimulatory
molecules, etc. (Schluns and Lefranc ¸ois, 2003; Surh and Sprent,
2005; Takada and Jameson, 2009). The cognate factors, on the
other hand, require sensing via the TCR, with the stimulatory
antigen being the best example (Obar et al., 2008; Smith et al.,
Even within these models, the relative contribution of either
category to T cell survival, especially in the context of CD4+
T cells, is far from clear. Early experiments suggested that
TCR-major histocompatibility complex (MHC) interactions were
quite critical for survival (Kirberg et al., 1997; Polic et al., 2001;
Takeda et al., 1996; Tanchot et al., 1997). Subsequent experi-
ments, however, controlling for factors such as cell proliferation
sary for CD4+T cell survival, and therefore could not be the crit-
ical determinant of their population control (Dorfman et al., 2000;
Grandjean et al., 2003).
A second set of experiments critical to understand peripheral
homeostasis is the behavior of CD4+T cells in lymphopenic
models. Under these conditions, otherwise quiescent naive
T cells can proliferate and differentiate, even in the absence of
their cognate antigen (Cho et al., 2000; Oehen and Brduscha-
Riem, 1999). In fact, this behavior has severe clinical ramifica-
tions, where aggressive immunopathology results from the
response of T cells in lymphopenic conditions generated during
Immunity 37, 735–746, October 19, 2012 ª2012 Elsevier Inc. 735
conventional tolerance induction (Brown et al., 2006; Schietinger
et al., 2012; Singh et al., 2006; Wu et al., 2004).
The common explanation for this lymphopenia-driven T cell
proliferation is that it reflects a response to an overabundance
of trophic factors that normally maintain peripheral homeostasis.
It occurs even in MHC-II deficient environments (suggesting that
the public factors alone are relevant) (Clarke and Rudensky,
2000; Grandjean et al., 2003), but it can only be blocked by
packing the host with cells of the same clonotype (suggesting
instead that cognate factors are critical) (Moses et al., 2003;
Troy and Shen, 2003). This paradox has nevertheless led to the
notion of ‘‘clonal competition,’’ which suggests that long-term
population control in the peripheral CD4+T cell compartment
is achieved by narrow competition between identical clones of
T cells (Hataye et al., 2006). However, it is very difficult to extrap-
olate such data from TCR transgenic model systems to a truly
polyclonal scenario. The frequency of any particular clonotypic
receptor in such a repertoire is likely to be exceedingly low,
making it difficult (but not impossible) to mediate such potent
effects (Quiel et al., 2011). In the absence of a high-resolution
functional dissection of natural polyclonal repertoires of T cells,
our understanding of these control mechanisms remains very
To address these issues, we designed a series of cellular
experiments exploiting the contrasting behavior of T cells in
lymphopenic or intact environments. After exhaustively elimi-
nating competition for public or conventional cognate factors
as the primary regulators of T cell frequency in these models,
latory component ab initio. In an unbiased in vivo screen, we
identified a specific T cell that was sufficient to constrain the
numbers of the self-reactive T cell and prevent its pathogenicity,
even in a lymphopenic environment. This control mechanism,
which we call ‘‘deletor’’ activity, involves specific T cells that
can impart a survival disadvantage onto self-reactive T cells,
even in the absence of the latter’s agonistic antigen, by
competing for a shared subthreshold self-ligand. These data
suggest that peripheral CD4+T cells are functionally organized
into relatively small colonies, as a result of the communal recog-
nition of specific subthreshold ligands. The control of population
dynamics primarily at the level of such colonies may have
evolved to reduce the risk of broad bystander repertoire loss
during each immune response.
Neighboring CD4+T Cells Limit the Pathogenic Potential
of Autoreactive T Cells
A dramatic illustration of the consequences of perturbing
homeostatic processes in the peripheral immune system is the
behavior of T cells in clinical or experimentally induced lympho-
penic environments. A model for dissecting these sequelae
involves adoptively transferring antigen-specific T cells to mice
expressing the target antigen. In experiments using 5C.C7
TCR-transgenic T cells responding to a self-antigen (transgeni-
cally expressed pigeon cytochrome C [PCC] under an MHC-I
promoter) in mice that are T cell deficient (PCC+, Cd3e?/?) or
intact (PCC+, with endogenous T cells), we have shown that
autoimmune arthritis develops only in the lymphopenic host
(Singh et al., 2006). The absence of disease in T cell-intact hosts
correlated with a slow ‘‘deletion’’ of the self-reactive T cells that
was not observed in the T cell-deficient hosts. This suggested
that host T cells are critical for effective control of the antigen-
specific response and can help decide between ‘‘disease’’ and
‘‘tolerance’’ in this context. We therefore designed a series of
experiments aimed at identifying the activity within a polyclonal
host T cell repertoire —which we will refer to as ‘‘deletor’’
activity—that elicits a phenotype akin to ‘‘clonal deletion’’ in
self-reactive T cells.
eral T cell population because self-reactive T cells in a PCC+,
Cd3e?/?host could be controlled by introducing new polyclonal
S1A and S1B available online). Deletors were also absent in
PCC+, Tcra?/?mice, implying that they are ab T cells rather
than gd T cells (Figure S1C). In fact, simply transferring a million
flow cytometry-sorted CD4+polyclonal ab T cells a day before
the 5C.C7 transfer was sufficient to trigger a 95% contraction,
whereas CD8+T cells had only a minimal effect (Figure 1A).
However, the deletor activity could not be further fractionated
within the CD4+pool based on the expression of CD44, CD25,
or FOXP3 (Figure 1B). This suggested that simply the presence
of an abundance of neighboring CD4+T cells might be sufficient
to control the population dynamics of antigen-specific T cells.
The Frequency of Autoreactive T Cells Is Unaffected
by Public or Clonal Competition
The ability of host CD4+ab T cells to limit the frequency of the
pathogenic CD4+5C.C7 T cells appeared to conform to existing
ideas regarding homeostasis of naive and memory T cells, i.e.,
a result of trophic competition between T cells of the same
Days after transfer
5C.C7 T cells
CD 4+ CD 44-lo CD25-
CD4+ CD44- hi
5C.C7 T cells
Figure 1. Neighbors Regulate the Frequency of Autoreactive T Cells
(A) Kinetics of expansion and contraction of PCC reactive 5C.C7, Rag2?/?
(open squares) compared to that in similar recipients who received 106poly-
clonal CD4+(red triangles) or CD8+(green circles) T cells 1 day earlier. Data
pooled from two experiments; n = 4 per time point.
(B) The number of 5C.C7 T cells in pooled lymph nodes and spleen of PCC+,
Cd3e?/?mice that were first injected with 0.5 (Foxp3-gfp+/?) or 1 3 106cells
(all other groups) of flow cytometry-sorted CD4+polyclonal T cell subsets (as
labeled on the x axis) and harvested 26 or 40 days later. In a one-way ANOVA
analysis, the control (0) group was significantly (p < 0.001) different from
all other groups, but the differences between subsets were not significant
(p > 0.05). Data are from two separate experiments with n = 3 or 4 per group.
Targeted Regulation of T Cell Homeostasis
736 Immunity 37, 735–746, October 19, 2012 ª2012 Elsevier Inc.
Supplemental Information includes seven figures, Supplemental Experimental
Procedures, andonetable andcanbefoundwiththisarticleonline athttp://dx.
We thank C. Chen and E. Chuang for assistance with experiments, C. Henry
andC.EigstiforFACS sorting, Q.Suand T.Myers formicroarrayhybridization,
P. Chappert for hearty discussions, and R. Germain and Y. Belkaid for
comments on improving the manuscript. This research was supported by
the Intramural Research Program of the NIH, NIAID.
Received: April 19, 2012
Accepted: August 13, 2012
Published online: September 27, 2012
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