Reduced Functional Avidity Promotes
Central and Effector Memory CD4 T Cell
Responses to Tumor-Associated Antigens
This information is current as
of June 15, 2017.
Stefano Caserta, Joanna Kleczkowska, Anna Mondino and
Rose Zamoyska
J Immunol 2010; 185:6545-6554; Prepublished online 3
November 2010;
doi: 10.4049/jimmunol.1001867
http://www.jimmunol.org/content/185/11/6545
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References
The Journal of Immunology
Reduced Functional Avidity Promotes Central and Effector
Memory CD4 T Cell Responses to Tumor-Associated
Antigens
Stefano Caserta,* Joanna Kleczkowska,* Anna Mondino,† and Rose Zamoyska*
T
he development of effective T cell memory is a hallmark
of the adaptive immune response and the goal of vaccination strategies. In recent years, a good deal has been
learned about the process that drives naive T cells to become effector and memory populations, and a number of genetic programs
relevant to the adoption of an effector or memory fate have been
described (1, 2). However, we still have a poor understanding of
the signaling events that initiate these decisions of fate, and many
questions remain about the relationships between the lineages that
give rise to effector and memory subpopulations. In particular,
debate has focused on whether there is a progressive linear differentiation path, such as naive T cell → effector T cell → memory
T cell (3), or whether effector or memory cells independently
differentiate directly from naive T cells (e.g., as a consequence of
an early asymmetric division) as has been suggested in some
studies (4). Clearly the initial encounter of TCRs on naive T cells
with Ag:MHC complexes (signal 1) and the interactions between
costimulatory molecules and their ligands on APCs (signal 2),
together with the surrounding inflammatory cytokine milieu (signal 3), play an important part in the subsequent differentiation
*Institute for Immunology and Infection Research, The University of Edinburgh,
Edinburgh, United Kingdom; and †Cancer Immunotherapy and Gene Therapy Program, Department of Oncology, San Raffaele Scientific Institute, Vita-Salute San
Raffaele Unversity, Milan, Italy
Received for publication June 7, 2010. Accepted for publication September 26, 2010.
This work was supported by Cancer Research UK Project Grant R40534 and by the
Medical Research Council UK. S.C. was initially supported by a fellowship from the
International Ph.D. Program in Molecular Medicine (Vita-Salute San Raffaele University).
Address correspondence and reprint requests to Rose Zamoyska, The University of
Edinburgh, West Main Road, EH9 3JT Edinburgh, United Kingdom. E-mail address:
[email protected]
Abbreviations used in this paper: BM, bone marrow; DC, dendritic cell; dLN, draining lymph nodes; Dox, doxycyclin; LACK, Leishmania analogue of the receptors of
activated C kinase; LN, lymph node; ndLN, nondraining lymph nodes; qPCR, quantitative PCR; TCM, T central memory; TE, T effector; Tet, Tetramer; WT, wild type.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001867
decisions of T cells. However, the biochemical consequences of
these interactions and an understanding of how they affect cellular
fate are poorly understood.
Efforts to identify signaling parameters downstream of the TCRs
that influence the differentiation of effector and memory fates have
largely concentrated on manipulating Ag dose or exposure and
monitoring subsequent outcomes. CD8 T cells seem to be able to
generate memory cells as efficiently to both low and high avidity
peptides (5) and following brief Ag exposure (6–8). For CD4 T cells
it appears that higher effective Ag doses (9–11) and longer Ag exposure times (12, 13) are required for efficient memory cell generation. It is unclear whether this apparent requirement for stronger
TCR signaling to generate CD4 memory T cells represents a fundamental difference in the differentiation potential between CD4
and CD8 T cells or whether it reflects differences in their activation
thresholds or functional avidity. The latter is influenced by a variety
of factors, such as the affinity of TCR-peptide:MHC binding, the
density of the ligand, and the distribution and composition of signaling molecules within the T cell, that dictate its sensitivity to
being triggered through the TCR. Alterations in any one or all of
these components can affect the differentiation outcome.
In contrast to acute viral and bacterial infections that are cleared
rapidly, tumors and persistent pathogens present an additional
problem to the development of effector and memory T cells. In
conditions of Ag persistence, the generation and maintenance of
memory-phenotype CD4 T cells is impaired, and repeated Ag
exposure drives terminal differentiation of precursors into shortlived effectors with a failure to accumulate systemic memorylike responses (14, 15). Thus, in contrast to in vitro studies
showing that higher degrees of TCR stimulation can favor differentiation to memory (16, 17), conditions of chronic Ag persistence in vivo can result in clonal exhaustion (18). Whether
exhaustion results from environmental pressures—such as the
nature of the APC, the contribution of TLR signals, or cytokine
availability in niches—or whether it is a result of intrinsic differences related to the functional avidity of individual T cell
clones is unknown.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
The effect of TCR signals on the differentiation of memory T cells is poorly defined. Conventional wisdom suggests that high-avidity
interactions are best for the selection of vaccine Ag candidates or T cell specificities for adoptive T cell therapy to stimulate robust
responses. However, in conditions of Ag persistence, high-avidity clones might exhaust and fail to form long-lived protective memory. We have manipulated the functional avidity of CD4 T cells by reducing expression of Lck, a key kinase involved in TCR triggering. Using a mouse model, we followed tetramer-positive T cells responding to a tumor Ag expressed by an adenocarcinoma. We
show that reducing the functional avidity increased effector–effector memory responses and improved the generation of selfrenewing, recirculating, tumor Ag-specific memory phenotype CD4 T cells. Moreover, such cells together with wild type CD8
T cells were better able to control tumor growth. Mechanistically, reducing Lck prolonged IL-2 production and cell turnover in
the central memory population while reducing expression of exhaustion markers in the face of chronic Ag. Our data indicate that,
in situations of persistent Ag challenge, generating T cells with reduced functional avidity may elicit more effective immune
responses. The Journal of Immunology, 2010, 185: 6545–6554.
6546
REDUCING AVIDITY IMPAIRS MEMORY CELL EXHAUSTION
To address the question of whether the functional avidity of
T cells can affect the formation and persistence of effector and
memory T cells, we used a mouse model in which we can alter
expression of Lck, the key proximal kinase involved in triggering the TCR, using a tetracycline-inducible transgene (19). We
have shown previously that reducing or removing Lck alters the
threshold at which cells can be triggered, thus reducing their
functional avidity (20). We examined how this reduction in sensitivity to stimulation affected the response to a persistent tumor
by following the response of tetramer+ cells from a TCR-b
transgenic mouse to a defined tumor Ag. Surprisingly, we found
that lowering the functional avidity favored the generation of
recirculating, self-renewing, multipotential, central memory-like
CD4 T cells together with a corresponding persistence of effector phenotype cells. In addition, these memory cells appeared to
be able to continuously seed the effector pool. In contrast, central
memory phenotype cells expressing endogenous Lck showed evidence of exhaustion and had correspondingly reduced effector
cell numbers. In addition, lower levels of Lck resulted in CD4
T cells that, with WT CD8 T cells, were better able to control
tumor growth. These data show that in situations of chronic Ag
persistence, a lowering of functional avidity can promote more
effective CD4 T cell responses.
described previously (24); 106 CD4+ LN-derived cells were cultured with
2.5 3 106 beads.
Materials and Methods
Two-tailed Student t test (unpaired) was used to assess statistically significant changes between groups.
16.2b transgenic mice on a BALB/c (H-2d) background express a transgenic TCR-b–chain (Vb4) specific for the immunodominant peptide
(Leishmania analog of the receptors of activated C kinase [LACK],
FSPSLEHPIVVSGSWD), derived from the Leishmania major LACK
protein and presented in the context of I-Ad, MHC class II molecules (21).
Lck1 Lck2/2 mice (19) were backcrossed to 16.2b mice to give 16.2bhom
Lck1homLck2/2 (H-2d) strains that were intercrossed with rtTA.ChomLck2/2
(H-2b, C57BL/6 background) to generate 16.2b Lck-inducible mice (H-2d/b).
16.2bhomLck1homLck2/2 were intercrossed with C57BL/6 mice to generate
16.2b H-2d/b control mice. Lck-inducible intercrosses were given doxycycline
(Dox) in food (1 mg/g) continuously from gestation. All mice were bred and
maintained in a specific pathogen-free facility at the Medical Research
Council National Institute for Medical Research under the U.K. Home Office
and local guidelines.
TS/A-LACK mouse mammary adenocarcinoma cells (4 3 105) (14)
were inoculated s.c. in the right flank. The axillary, brachial, and inguinal
lymph nodes (LNs) draining the site of tumor cell injection were referred
as to tumor-draining LNs (dLN). The axillary, brachial, and inguinal LNs
derived from the contralateral flank were referred to as tumor nondraining
LNs (ndLN). Tumor growth was monitored every other day by measuring
with a metric caliper. Tumor diameters were measured in three dimensions,
and their volumes were calculated by multiplying the diameters (in cubic
millimeters). BrdU (1.2 mg per mouse) was injected i.p and added to the
drinking water (0.8 mg/ml) 4 d before analysis. BrdU incorporation was
determined as described previously (22).
Mixed bone marrow (BM) chimeras were made by reconstituting 500rad irradiated (1 d prior to transplant) BALB/c Rag2/2 mice i.v. with 107
T cell-depleted BM pools of 1:9 16.2b wild type (WT) (H-2d):16.2b Lckinducible (H-2b/d) cells. Reconstituted mice were fed a Dox diet, and peripheral reconstitution was assessed by screening blood. For adoptive
transfer experiments, naive H-2d/b CD8 T cells were purified by positive
selection with anti-CD8PE and anti-PE conjugated MACS MicroBeads
(Miltenyi Biotec, Auburn, CA) and transferred i.v. at day 0, 2, 10, 12 (106
cells per mouse).
Mouse T cell primary cultures
LN cells were labeled with the fluorescent dye CFSE as described (23).
Cells were cultured at 37˚C in complete medium (RPMI 1640, 10% FBS,
100 U/ml penicillin, 100 U/ml streptomycin, and 2.5 3 1025M 2b-ME;
Invitrogen, Carlsbad, CA) for the number of days specified in each experiment. Dox (2 mg/ml) (Sigma-Aldrich, St. Louis, MO) was added
in vitro every 2 d. Anti-CD3 (145-2C11, 0.01 mg/ml) and anti-CD28
(37.51; 5 mg/ml) mAb were coated on plates for 1 h at 37˚C in PBS,
and the wells were washed twice before adding cells. LACK-specific tetramers plus anti-CD28 Ab were coated onto Latex beads (Invitrogen) as
I-Ad/LACK multimer staining was performed as described (14). Cells
were labeled with PE-TexasRed- (Caltag Laboratories, Burlingame, CA),
PE- APC-, or PerCP-labeled Abs: anti-CD4, anti-CD44, anti–H-2b, antiCD62L, anti-CD8a, anti-CD11b, and anti-B220 mAb (BD Pharmingen,
San Diego, CA; eBioscience, San Diego, CA). TO-PRO-3 (1 nM final
concentration; Molecular Probes, Invitrogen) was added to the sample just
before FACS analysis. Events were collected (3 3 105 CD4+ or at least 103
CD4+ I-Ad/LACK+) by excluding all the anti-CD11b+, anti-B220+, antiCD8a+, and TO-PRO-3+ events. Cells were stained with directly conjugated Abs for 15 min at 4˚C, washed, and acquired on FACSCalibur, LSR
I, or LSR II flow cytometers (BD Biosciences, San Jose, CA).
Quantitative PCR analyses
Highly purified (.97%) T central memory (TCM) and T effector (TE) were
obtained by sorting with MOFLO-1 cell-sorters (Beckman Coulter, Brea, CA).
Cell pellets (1 3 107) were lysed for RNA extraction using the RNeasy Mini
Qiagen (Qiagen, Valencia, CA) protocol. RNA was reverse transcribed to
cDNA using SuperScript III First-Strand Synthesis System for RT-PCR
(Invitrogen). Quantitative PCR (qPCR) reactions were performed in the presence of TaqMan primers specific for mouse Hprt, Klrg1, Pdcd1 (PD-1), Prdm1
(Blimp1), and Casp3 (Applied Biosystems, Foster City, CA) and normalized to
Hprt expression levels for each sample.
Statistical analysis
Results
Low expression of Lck reduces the functional avidity of T cells
We used an experimental system in which we could follow the
response of CD4 T cells to a model tumor-associated Ag. 16.2b
mice are transgenic for a rearranged TCR-b–chain isolated from
a CD4 T cell clone specific for a Leishmania major Ag, LACK
(25). To alter the functional avidity of the responding T cells,
mice expressing a tetracycline-inducible Lck transgene (19) were
backcrossed to 16.2b mice (16.2b/Lck1/LckKO mice) and subsequently intercrossed with mice expressing the Tet-ON transactivator, rtTA (19). These mice were on an endogenous Lck2/2
background, and expression of Lck in the offspring was exclusively induced and maintained by the inclusion of Dox in food
(16.2b LckON) or, when required, Lck expression could be
extinguished by withdrawal of Dox for at least 7 d (16.2b
LckOFF). Because the Lck-inducible mice were of MHC H-2d/b
haplotype and heterozygous for the TCRb transgene, control mice
were generated by intercrossing 16.2b/Lck1/LckKO mice with
C57BL/6 mice. Although heterozygous for endogenous Lck, these
controls behaved similarly to homozygous 16.2b animals (data not
shown) and are hereafter referred to as WT 16.2b mice.
In the context of a polyclonal TCR-a repertoire, LACK-specific
T cells occur at a frequency of ∼0.5% in unchallenged 16.2b
control animals, which is higher than that found in naive BALB/c
animals and can be followed by staining with specific peptide:
MHC multimers (Fig. 1A). The LACK tetramer was shown previously to have essentially no reactivity with irrelevant T cells,
because staining was undetectable on DO11.10 TCR transgenic
cells (24). The frequency of LACK+ clonotypic T cells in 16.2b
LckON mice was comparable to WT controls, and they had a
naive, CD44low phenotype (Fig. 1A). As described previously
(19, 23), the amount of Lck in naive T cells from LckON mice is
∼10–20% of WT levels and is found primarily in association with
CD4 (20). Lck prevents CD4 internalization into endosomes and
thus contributes to the maintenance of CD4 surface expression
(26). Consequently, CD4 surface expression was lower in LckON
cells (Fig. 1A) and acted as a convenient indicator of Lck levels in
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Mice and tumor lines
Flow cytometry
The Journal of Immunology
6547
these cells (20). The proportions of naive and memory cells in
nonimmunized 16.2b LckON mice were similar to those found in
WT 16.2b mice (Fig. 1A, right panel).
The reduced expression of Lck made 16.2b LckON cells less
responsive to anti-CD3 plus anti-CD28 stimulation than were
WT cells, as measured by upregulation of early activation markers
(Fig. 1B) or proliferation and IL-2 production (20). Notably,
without provision of Dox in vitro, LN cells from LckON mice
failed to proliferate to anti-CD3 stimulation unless anti-CD28
costimulation was provided, whereupon some cells were triggered to respond (Fig. 1C). Supplementation of Dox greatly enhanced the frequency of LckON cells that were triggered, and
more CFSE dilution was apparent in this population than in the
WT counterparts by day 7. The increased proliferation of LckON
cells was due in part to these cells being more resistant to activation-induced cell death (see below), but WT cells can be
inhibited slightly by Dox in the cultures as seen at day 7.
Ag-specific stimulation of 16.2b LckON cells with clonotypic
I-Ad/LACK multimers plus anti-CD28 showed an absolute requirement for Dox to detect proliferation from this low initial
frequency of cells (Fig. 1D). In the presence of Dox, a substantial
proliferative response was observed from LACK-specific LckON
cells, and TO-PRO-3 staining revealed that far fewer of the
proliferating LckON cells died compared with WT cells (9 and
33%, respectively). Therefore, low expression of Lck in LckON
cells effectively reduced the functional avidity of these cells, as
demonstrated by the raised activation threshold of naive T cells
and their increased dependency on costimulation for activation. In
addition, low levels of Lck favored survival of the proliferating
cells.
Priming in the presence of low Lck favors systemic
redistribution and self-renewal of memory-like cells
We examined how reducing the functional avidity of T cells might
affect their ability to form effector cells, long-term central
memory cells, or both in vivo. 16.2b transgenic mice have been
used previously in tumor studies (14) using the TS/A-LACK adenocarcinoma cell line. TS/A-LACK cells are TS/A cells stably
transfected with LACK protein, which is expressed intracellularly
and functions as a model tumor-associated Ag (14). After s.c.
injection of TS/A-LACK tumor-cells, 100% of 16.2b animals
developed a solid tumor localized in one flank, and it did not
metastasize. Because 16.2b transgenic mice, compared with WT
mice, have an increased frequency of LACK-specific CD4 T cells,
it was possible to trace the endogenous CD4 T cell responses to
the LACK Ag using I-Ad/LACK multimers. In this system, Ag
presentation to CD4+ cells relies on endogenous APCs, because
the tumor cells themselves do not express MHC class II. Despite
the lack of direct Ag presentation, CD4 T cells were shown to play
a role in controlling growth of the tumor, because tumor size was
significantly increased after treatment of tumor-bearing mice with
anti-CD4 Abs in vivo (14).
In 16.2b WT mice 12 d after tumor cell injection, when tumor
size was relatively small, LACK-specific CD44+ cells expanded
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FIGURE 1. LckON naive CD4 T cells have
a higher stimulation threshold in vitro. A, Staining
of LN cells ex vivo shows few I-Ad/LACK tetramer+ (Tet+) T cells in BALB/c mice, whereas
similar frequencies of Tet+ cells are present in
16.2bWT and LckON mice (top panel) and they
have a naive CD44lo phenotype (bottom panel).
Numbers in the quadrants of 16.2bWT and LckON
mice are the mean frequency 6 SD from 10 animals. B, LckON LN cells cultured with a titration
of anti-CD3 plus 5 mg/ml anti-CD28 require more
stimulation to achieve equivalent CD69 upregulation to WT cells. C, Proliferation of LckON CFSElabeled cells requires CD28 costimulation and is
enhanced by supplementation of Dox every 2 d to
cultures in vitro. D, LN cells from LckON mice
proliferate to tetramer plus aCD28-coated beads
only in the presence of Dox and show less cell
death (TOPRO-3+ staining) after stimulation for
7 d. Data are representative of three independent
experiments.
6548
REDUCING AVIDITY IMPAIRS MEMORY CELL EXHAUSTION
by ∼20-fold and were more highly represented near the site of
tumor growth (i.e., the tumor-draining LN, dLN; Fig. 2A). In
contrast to the naive phenotype, CD44lowCD62Lhi, of control cells
from nonimmunized mice (Fig. 3A), the day 12 responding cells
were largely of a TE-effector memory, CD44hiCD62Llow, phenotype (hereafter collectively referred to as TE; Figs. 2B, 3B, upper
panel) and were also detected to a lesser extent in a distal lymphoid site (usually the contralateral flank from the localized site of
tumor development, ndLN). In addition to a TE population, d12
tumor-bearing WT mice had a distinct population of CD44hi
CD62Lhi cells that were distributed approximately equally between the dLN and ndLN (Figs. 2C, 3B, upper panel). The ability
to redistribute systemically around lymphoid organs is one of the
hallmarks of central memory T cells (14, 27), and this together
with the phenotype suggested that these CD44hiCD62Lhi cells
were a TCM population. By day 19, when the tumor load had
increased substantially, there was an ∼5-fold drop in the total
number of LACK+CD44hi cells in the dLN compared with that
found at day 12, whereas the ndLN numbers of LACK+CD44hi
cells reverted to that found in naive animals (Fig. 2A). The
remaining LACK-specific cells at day 19 were phenotypically
CD44hiCD62Llo effector cells mainly confined to the draining LN
(Figs. 2B, 3B, lower panel), and there was almost a complete
absence of TCM cells in either dLN or ndLN, both in numbers
(Fig 2C) and as a percentage (Fig. 3B, lower panel), consistent
with exhaustion of the tumor specific memory response.
Challenge of 16.2b LckON mice with tumor resulted in a
comparable LACK-specific response at day 12 when tumor burden was low. Similar to WT 16.2b mice, there was an increase in
LACK+CD44hi cells in both the dLN and ndLN at this time (Fig.
2A) with TE and TCM detectable at both sites (Fig. 3C,
upper panel). Remarkably, by day 19 this situation altered considerably and, in contrast to WT mice, we observed sustained
accumulation of LACK-specific CD44+ cells in both DLN and
ndLN of LckON mice (Figs. 2A, 3C, lower panel) regardless of
the increased tumor load, which was a similar volume in both
types of mice (data not shown). Moreover, both TE and TCM
phenotype cells accumulated (Fig. 3C, lower panel). Both TE
(Fig. 2B) and TCM (Fig. 2C) were found in significantly higher
numbers in the dLN and ndLN compared with WT animals.
Therefore, reducing the functional avidity by lowering the availability of Lck enabled protection against the contraction of the
response and its restriction to the lymphoid site most proximal to
tumor Ag persistence. Moreover, low Lck-containing, LckON
mice maintained a population of TCM cells that both persisted and
recirculated and may have continuously fed conversion into the
effector pool.
The expansion and persistence of TCM in tumor-bearing LckON
mice was not particular to LACK-specific T cells as total CD44hi
T cells were increased (Fig. 2D). It has been shown previously that
transfer of comparable CD4 T cells from tumor-bearing animals
was protective against tumor challenge, indicating that the tumor
stimulates multiple specificities in addition to the 16.2b epitope
(28). Subdivision of total CD44hi T cells into CD62Lhi (TCM) or
CD62Llo (TE) showed that WT and LckON mice accumulated
similar numbers of both when compared at day 12 after tumor
development. At day 19, only LckON mice continuously accumulated memory-like cells despite the chronic Ag exposure. Thus,
in animals expressing WT Lck, all tumor-responding CD4 T cells
were consumed over time in the presence of persistent Ag,
whereas they were continuously maintained in low Lck levels. A
similar preference for maintenance of both TE and TCM populations in the presence of tumor was found for mice that lacked
Lck altogether (LckOFF mice). Although fewer total tumorresponsive cells were generated during this time, they continued to
accumulate rather than decline like WT cells, indicating that
signals through Fyn were sufficient to drive differentiation of both
these subsets of cells.
Low levels of Lck sustain proliferation in the face of chronic Ag
It was possible that the different numbers of memory-like cells
found in WT and LckON mice reflected diverse proliferative
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FIGURE 2. Tumor-specific LckON CD4 T cells do not exhaust from Ag persistence. Absolute numbers of subpopulations of Ag experienced CD44+
CD4 T cells from LN draining the site of tumor (dLN) or from the contralateral flank (ndLN) are presented for gated LACK-Tet+ cells (A–C) and for all
remaining CD44hi cells (D) on day 12 (filled circles) and day 19 (open circles) after tumor inoculation. Groups are from naive mice (no tumor challenge).
LckWT mice (WT), LckON mice (ON) and LckOFF mice (OFF) which had been taken off a Dox diet at least 10 d prior to tumor challenge. Gates used for
defining TE (B) and TCM (C) are shown in Fig. 3. Statistically significant groups are indicated (*p , 0.05; **p , 0.005). Data are representative of at least
three independent experiments of two to five mice per group.
The Journal of Immunology
6549
capacities of cells primed in the presence of high or low levels of
Lck. It was shown previously (14) that the peak of the response to
this tumor in WT BALB/c mice is at ∼4 d after tumor challenge.
Similar numbers of responding cells were recovered in WT and
LckON mice at ∼6–7 d after tumor challenge (Fig. 4A); to assess
their proliferation, we measured the frequency of BrdU+ CD4+
T cells in the dLN and ndLN of tumor-bearing mice after continuous BrdU administration for the final 4 d before analysis.
Seven days after tumor cell innoculation, comparable frequencies
of BrdU+ CD4+ T cells were found in WT and LckON mice (Fig.
4B), indicating that the rate of cycle between the two populations
was similar at the start of the response. By day 14, many fewer
WT cells had incorporated BrdU (4% compared with 13% at day 7
in the dLN), whereas the proliferation of LckON cells at day 14
was unchanged from day 7 (Fig. 4C). This sustained proliferation of LckON cells was dependent on Lck expression, because
proliferation decreased in a cohort of mice that were taken off a
Dox diet at day 7 and analyzed at day 14. Therefore, Lck being
expressed at low levels supported long-term turnover of Ag-
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FIGURE 3. Distribution of TE- and TCM-like, tumor-specific CD4 T cells in mice expressing different
levels of Lck. A, FACS analysis shows that Tet+ cell in
16.2b LckON LNs are predominantly naive for two
animals taken at day 12 (first two panels, left) and d19
(last two panels, right) time points. B, In WT mice,
populations of Tet+ TCM (CD44hiCD62Lhi) and TE
(CD44hiCD62Llo) are detectable in dLN and ndLN at
d12 (upper panel), but by day 19 (lower panel) both
populations have largely disappeared from ndLN and
only TE are found in the dLN. C, In contrast, in
LckON mice both TCM and TE increase between day
12 (upper panel) and day 19 (lower panel) and are
detectable in dLN and ndLN.
responsive cells and mitigated against the decline in self-renewing
capacity, which occurred in cells expressing WT Lck. In naive
mice, rates of BrdU incorporation were similar in WT and LckON
mice (Fig. 4D), indicating that the background turnover rate of
homeostatic CD4 memory cells is similar in these animals in the
absence of overt antigenic stimulation.
It has been shown that IL-2 confers a survival advantage on
CD4 T cells (29) and is important for T cell proliferation;
therefore, we asked whether there was a difference in the production of IL-2 after immunization in vivo between LckON and
WT animals. We immunized mice with LACK peptide pulsed
dendritic cells (DCs) so that we could recall Ag-specific IL2 producing cells at the peak of the response at day 5, as shown
previously (27). Indeed, at this time there were substantially
more peptide-specific CD4 cells with the potential to produce
IL-2 in LckON mice (Fig. 5). Similar results were found at day
19 in tumor-bearing animals with more IL-2–expressing cells
found in LckON mice (data not shown). Moreover, in LckON
tumor-bearing mice we found increased numbers of cells ex-
6550
REDUCING AVIDITY IMPAIRS MEMORY CELL EXHAUSTION
γ
FIGURE 4. Sustained proliferation of LckON CD44hi CD4 T cells in
tumor-bearing mice. A, Numbers of Tet+ CD44hi cells recovered at days 7,
14, and 21 after tumor challenge of WT and LckON mice. B, Mice were
given BrdU from day 3 of tumor challenge until day 7, and similar proliferation rates were seen among CD44hi cells in WT and LckON animals.
C, By day 14 of tumor challenge, an equivalent 4-d BrdU pulse labeled
many more dividing cells in LckON compared with WT mice. Dox
withdrawal at day 7 reduced proliferation, indicating that proliferation
relies on Lck expression. D, LckON and WT naive mice labeled for 4 d
show equivalent BrdU incorporation. Data are representative of two independent experiments.
pressing both CD25 and CD127 (data not shown), suggesting
that more IL-2–responsive cells were able to enter the memory
pool in these animals.
Lck levels shape the phenotype of memory cells in a
T cell-intrinsic manner
In LckON mice, it was possible that T cells expressing low levels of
Lck accumulated after priming, because of differences in environmental factors rather than as a consequence of intrinsic difference in the way in which TCR signals were integrated. If, for
example, WT cells that were stimulated to become terminally
differentiated effector cells were able to kill Ag-bearing APCs or
engaged in fratricide or production of cytokines that affected
the T cell differentiation or survival, it might negatively affect
the generation of TCM. Alternatively, the growing tumor might
dynamically change in the presence of different Ag-specific responses, in terms of clonality or secretion of suppressive cytokines, so that the emerging Ag-specific CD4 T cell population
might be differentially affected in animals expressing different
levels of Lck. To investigate whether cell-intrinsic versus environmental factors played a role, we generated mixed BM chimeras
from donor 16.2b WT and 16.2b LckON mice. In this instance,
homozygous 16.2b WT (H-2d) donors were used to distinguish
them from LckON H-2bxd cells; because these were BM chimeras,
the T cells arising from the two genotypes were tolerant of one
another. The BMs were mixed in a 9:1 LckON:WT ratio, to generate approximately equal representation of the two cell types in
the periphery, and injected into irradiated BALB/c Rag2/2 hosts
(Fig. 6A). After 12 wk in the presence of Dox, reconstitution was
confirmed by analysis of peripheral blood, and recipients with
similar representation of both genotypes of T cells (Fig. 6A) were
injected with TS/A-LACK tumor cells.
Staining with anti–H-2Kb Ab permitted identification of the
phenotype of LACK-specific CD4 T cells expressing normal or
low Lck levels at day 19 of tumor development in the same animal
(Fig. 6B). Compared with naive controls, TS/A-LACK tumorbearing chimeras accumulated LACK-specific CD4 T cells with
TCM and TE phenotypes at high frequency (Fig. 6B, compare top
left [naive] with lower left [tumor-bearing] panel). As before,
LACK-specific CD4 T cells derived from cells expressing normal
levels of Lck mainly differentiate toward a TE rather than a TCM
phenotype (∼6:1 ratio, gated WT) (Fig. 6B, lower middle panel).
In contrast, LACK-specific CD4 T cells derived from cells
expressing lower levels of Lck maintained both TE and TCM
phenotypes in approximately equal proportions (Fig. 6B, lower
right panel), indicating that CD4 T cells expressing different
levels of Lck behaved in the chimera as they did in separate
animals.
Once again, injection of BrdU 2 d prior to analysis (Fig. 6C)
showed that T cells from WT progenitors proliferated less by day
19 than T cells derived from LckON cells, confirming that cells
expressing low levels of Lck do not exhaust their self-renewal
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FIGURE 5. DC vaccination stimulates more IL-2–producing cells in
LckON mice than in WT mice. Mice were challenged with LACK peptidepulsed autologous BM-derived DCs 5 d before analysis. LN cells were
restimulated for 6 h with LACK peptide-pulsed splenic APCs in the
presence of brefeldin A before staining with tetramer and CD44 (top
panel) or intracellular staining for IL-2 and IFN-g (bottom panel). LckON
mice showed a higher proportion of IL-2–staining cells than WT mice,
indicating preferential expansion of this population in conditions of low
Lck expression.
The Journal of Immunology
6551
potential despite chronic Ag persistence. As before, long-term proliferative responses were dependent on continual expression of
low Lck, because switching off Lck expression 14 d after tumor
injection (ON dox → OFF dox) reduced turnover of LckON cells.
Cells with reduced Lck show less evidence of exhaustion
It was likely that the disappearance of the WT TCM population was
due to exhaustion in the face of chronic Ag exposure. We reasoned
that, even at early time points when recirculating CD4 T cells were
found at comparable levels in the lymphoid organs of both WT and
LckON tumor-bearing animals, WT cells with a TCM phenotype
might already differ in the expression of exhaustion markers as
a prelude to their early disappearance in response to systemic
chronic Ag stimulation. Expression of a number of markers that
correlate with exhaustion have been described, mainly but not
exclusively in CD8 effector T cells, including CD127, KLRG1,
PD-1, and Blimp-1. Thus, high expression of CD127 together with
low expression of KLRG-1 was inversely correlated with exhaustion in CD8 T cells (30), whereas PD-1 was shown to be
upregulated early upon manifestation of exhaustion (31, 32). In
addition, Blimp-1 was shown to be upregulated in exhausted CD8
T cells in vitro and in vivo (33, 34) and to be upregulated late in
CD4 T cell differentiation (35, 36).
TCM phenotype cells from WT and LckON mice 11 d after
tumor innoculation were sorted, and mRNA was analyzed by qPCR
for expression of exhaustion markers. We consistently found that
LckON TCM cells expressed significantly less KLRG1, PD-1, and
Blimp1 than did WT cells (Fig. 7A), suggesting that although
phenotypically TCM, WT cells are more terminally differentiated
and prone to exhaustion than their LckON counterparts. Finally,
we checked the expression levels of Caspase-3, a terminal effector protease required in apoptosis, and found that TCM cells from
WT animals express higher levels of Caspase-3 mRNA than do
LckON TCM-like cells, suggesting that cells that are chronically
activated in the presence of higher levels of Lck are associated
with activation-induced cell death.
CD127 expression has been linked to the survival of CD4+
memory cells (37), and in CD8 effector cells CD127 expression
has been reported to indicate that these cells are destined to enter
the TCM pool (38). However, no difference was found in the
amount of CD127 on cells from WT or LckON mice with tumors
either at early (11 d) or later (20 d) time points (data not shown).
In addition, we monitored Bcl-2 expression and there was no
difference in the proportion of CD44+ cells that expressed Bcl-2
nor in the levels of expression, either by intracellular staining or
by qPCR (data not shown).
LckON CD4 T cells help control tumor growth
Tumor growth can be controlled by CD8 CTLs that mediate direct
cytolytic activity against tumor cells. In the TS/A-LACK adenocarcinoma model, the tumor cells are not direct targets for CD4mediated killing, because they lack MHC class II expression
and their parent line TS/A relies on CD8+ T cells to be rejected
(39). However, treatment of tumor-bearing mice with anti-CD4
Ab was shown to result in increased tumor bulk (14), whereas
adoptive transfer of tumor-specific CD4 cells conferred partial
protection (28), indicating that CD4 helper cells were involved in
restricting tumor growth. The 16.2b rearranged Vb4 TCR transgene came originally from a CD4+ cell and may skew the repertoire of CD8 cells that develop in these animals. Therefore, to
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FIGURE 6. Lck levels shape the phenotype of memory cells in a T cell-intrinsic manner. A, Schematic of the experiment showing that mixed BM
chimeras were reconstituted with 16.2b WT (H-2d) and 16.2b LckON (H-2b/d) cells and kept on Dox diet. Chimeras were checked for reconstitution and
injected with tumor. One group of mice had Dox removed from their diet at d7, the remainder stayed on Dox diet. All mice were given BrdU 2d before
sacrifice at day 19. B, Total CD4 cells (left panel) from naive (upper) or tumor injected mice (lower) are shown stained for TCM (CD44hiCD62Lhi) and TE
(CD44hiCD62Llo). The right two panels show gated WT or LckON cells from the same animals, which reveals that the TCM (CD44hiCD62Lhi) derives
mainly from LckON donor cells. The ratio of TE:TCM was 1.26 6 0.43 SD for LckON cells and 6.4 6 2.8 for WT cells from seven mice per group (p ,
0.0005). C, CD4+ LckON cells proliferate more than WT cells from an individual tumor-bearing chimera (top panel). Average BrdU-positive cells were
10 6 2% for LckON cells compared with 6.7 6 1% for WT for seven mice per group (p , 0.002). This depends on continuous Lck expression as
proliferation declines when Lck is switched off (lower panel). Data are representative of two independent experiments with four to six mice per group.
6552
address whether the CD4 memory cells that develop in tumorbearing LckON animals had beneficial antitumor effects, we infused naive polyclonal CD8 T cells into tumor-bearing mice and
looked for control of tumor growth.
16.2b LckON mice that received infusions of naive CD8 T cells
had smaller tumors compared with WT 16.2b mice and 16.2b
LckON mice that did not receive naive CD8 T cell infusion (Fig.
7B). Although the control of tumor growth tended to be transient,
comparison of LN cells 1 wk after the final CD8 T cell infusion
showed there was a population of CD44hi CD8 T cells clearly
visible in LckON mice, which was absent from WT controls, indicating that greater numbers of Ag-reactive CD8 T cells were
maintained in the LckON recipients (Fig. 7C). A substantial
proportion of these CD44hi cells originated from the infused cells
as, because ∼41 6 3% were negative for the Vb4 transgene
compared with 7 6 0.2% in the WT mice (data not shown). These
data indicate that LckON CD4 T cells exerted better functional
helper activity on the infused CD8 T cells to control tumor growth.
Discussion
The role of T cell avidity in the generation of effector and
memory cells was addressed in this study by manipulating an
intracellular kinase, Lck. We have shown previously that, although
primary T cells can respond to TCR ligation in the absence of Lck if
they express the related Src-family member Fyn, the threshold of
triggering is raised substantially (20). In this study, we showed that
the absence of Lck in LckOFF mice did not preclude formation of
phenotypically distinct TCM and TE subpopulations in response
to Ag exposure, but their numbers were limited by their impaired
ability to expand. WT levels of Lck resulted in robust early expansion of TCM and TE, but in time these numbers reduced in the
face of persistent Ag. In this context, neither too much nor too
little signal seemed to be achieved with ∼10% of normal Lck
levels in LckON mice, which resulted in continuous accumulation
of both TCM and TE with specificity for tumor-associated Ag.
In the interest of clarity, we defined the CD44hiCD62Llo population of Ag-responding cells as TE, given that tumor Ags were
continually present, and they preferentially accumulated in the
Ag-draining LN over time; however, this population could contain
both effector cells and effector memory cells. Similarly, in mouse
but not human studies, long-term central memory cells are generally defined as persisting in the absence of Ag, but because the
tumors were not rejected we did not follow this population for
a long term. However, LACK-specific cells with a CD44+CD62L+
phenotype were generated after peptide-DC vaccination in both
16.2b and WT mice and were protective in the latter (27), suggesting this phenotype demarcates bona fide memory cells. Moreover, it has been suggested that long-lived central memory cells
can be programmed from as early as the first cell division after
Ag encounter (4), and our data are compatible with their early
generation. Although our data do not directly address the relationship between TCM and TE that were generated to tumor Ags,
it is clear that both subpopulations were detectable at similar
initial frequency regardless of the strength of signal (i.e., whether
Lck was high, low, or absent). Therefore, our data are not compatible with models that suggest that the signal strength per se
directly influences whether a cell becomes a TE or TCM. In WT
mice, decline of TCM by day 19 of tumor growth was mirrored by
a drop in TE, which is consistent with either an early asymmetric
fate decision (4) or a “fate commitment with progressive differentiation” type model of effector cell generation from a concomitantly generated, cycling TCM pool (3). In addition, the continual
presence of both TCM and TE phenotype cells in LckON animals
suggested that the former might be continuously seeding the latter.
We have shown previously that Lck influences multiple signaling pathways downstream of TCR engagement, but that not all
pathways are equally dependent on Lck (20). For example, activation of PLCg1 and Ca2+ mobilization were much more dependent on Lck than activation of the ERK/MAPK pathway. One
consequence of these perturbations in signaling was that the
production of IL-2 was reduced in LckON and LckOFF cells (20).
More recently, we have found that the kinetics of IL-2 production
is also influenced by reducing Lck in CD8 T cells (J. Kleczkowska, unpublished data) so that although less IL-2 is produced,
production continues for much longer, up to 48 h in vitro. Generally, IL-2 production from naive T cells is transient (40), because the cytokine stimulates a negative feedback loop upon
binding the IL-2R that shuts off IL-2 transcription (41). Therefore,
lower levels of cytokine that might not activate the feedback loop
could sustain IL-2 production, and prolonged IL-2 availability
has been shown to promote sustained expansion, certainly for
CD8 T cells (42, 43). In addition, IL-2 was shown to enhance
CD4 T cell survival (29) and memory generation (44). Consistent
with this finding, we found an increased proportion of LACKspecific cells expressing intracellular IL-2 in LckON animals after
DC vaccination and in tumor-bearing mice. Moreover, there were
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FIGURE 7. LckON cells show less evidence of exhaustion in the presence
of chronic Ag and promote control of tumor growth in the presence of infused CD8 T cells. A, RNA was extracted from FACS sorter purified TCM
from WT and LckON tumor-bearing mice and reverse transcribed for qPCR
analysis for expression of genes implicated in cell exhaustion. Expression of
Klrg1, Pdcd1 (PD-1), Prdm1 (Blimp-1), and Casp3 (Caspase 3) as indicated
from three biologic replicates is upregulated in WT cells significantly more
than in LckON cells (*p , 0.05; **p , 0.005). B, Tumor mass (in cubic
milliliters) at day 19 of tumor growth is substantially reduced in 16.2b
LckON compared with WT mice that had received adoptively transferred
CD8 T cells from naive WT donors on days 2 and 9 after tumor innoculation
(*p , 0.02) or from LckON mice that did not receive CD8 T cells. C,
Histograms show that the percentage of CD44+ CD4 and CD8 T cells in both
dLN and ndLN of recipient LckON mice is higher than from WT recipients
1 wk after the last adoptive transfer with purified CD8 T cells. Data are
representative of two independent experiments.
REDUCING AVIDITY IMPAIRS MEMORY CELL EXHAUSTION
The Journal of Immunology
Acknowledgments
We thank Valentino Parravicini and Peter Tomlinson for help with the
experiments, Ben Seddon for advice with the experiments, the Zamoyska
laboratory for general discussions, and Paul Travers and David Gray for
critical comments on the manuscript.
Disclosures
The authors have no financial conflicts of interest.
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