Download CD8 -Mediated Survival and Differentiation of CD8 Memory T Cell

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Fig. 4. Mechanism of peptide splicing. (A) Model of the peptide splicing reaction. (B) Various synthetic
peptides were combined in a pairwise manner and incubated with 20S proteasomes. Digests were tested
for recognition by CTL 14. Mass spectrometry confirmed the presence of RTKQLYPEW in the digests
recognized by the CTL and its absence in the others. Ac-QLYPEW, N-␣-acetylated peptide QLYPEW.
alytic. Rather, it is catalyzed by the proteasome and
therefore takes place during protein degradation.
Peptide identification efforts have provided
many examples of antigenic peptides that do not
simply correspond to fragments of conventional
proteins, but rather result from aberrant transcription, incomplete splicing, translation of alternative or cryptic open reading frames, or posttranslational modifications (11–16). Peptide
splicing is another mechanism that increases the
diversity of antigenic peptides presented to T
cells. It represents a new aspect of the proteasome function in antigen processing.
References and Notes
1. K. Hanada, J. W. Yewdell, J. C. Yang, Nature 427, 252
(2004).
2. K. L. Rock, A. L. Goldberg, Annu. Rev. Immunol. 17,
739 (1999).
CD8␣␣-Mediated Survival and
Differentiation of CD8 Memory T
Cell Precursors
Loui T. Madakamutil,1 Urs Christen,1 Christopher J. Lena,1
Yiran Wang-Zhu,1 Antoine Attinger,1 Monisha Sundarrajan,1
Wilfried Ellmeier,2* Matthias G. von Herrath,1 Peter Jensen,3
Dan R. Littman,2 Hilde Cheroutre1†
Memory T cells are long-lived antigen-experienced T cells that are generally
accepted to be direct descendants of proliferating primary effector cells.
However, the factors that permit selective survival of these T cells are not
well established. We show that homodimeric ␣ chains of the CD8 molecule
(CD8␣␣) are transiently induced on a selected subset of CD8␣␤⫹ T cells
upon antigenic stimulation. These CD8␣␣ molecules promote the survival
and differentiation of activated lymphocytes into memory CD8 T cells. Thus,
memory precursors can be identified among primary effector cells and are
selected for survival and differentiation by CD8␣␣.
The majority of T cells responding during a
primary immune response subsequently undergo programmed cell death. However, a
fraction of activated T cells survive and
differentiate into long-lived memory T
cells (1). What mechanisms mediate the
590
selective survival of these cells? To address
this question, we first must identify those
effector T lymphocytes that will differentiate into memory cells.
The homotypic form of CD8 that uses
the ␣ chain of the molecule (CD8␣␣) ap-
3. S. Morel et al., Int. J. Cancer 83, 755 (1999).
4. G. J. Adema, A. J. de Boer, A. M. Vogel, W. A. M. Loenen,
C. G. Figdor, J. Biol. Chem. 269, 20126 (1994).
5. Materials and methods are available as supporting
material on Science Online.
6. Single-letter abbreviations for the amino acid residues are
as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H,
His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg;
S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
7. S. Kageyama, T. J. Tsomides, Y. Sykulev, H. N. Eisen,
J. Immunol. 154, 567 (1995).
8. D. J. Irvine, M. A. Purbhoo, M. Krogsgaard, M. M. Davis,
Nature 419, 845 (2002).
9. M. Groll, R. Huber, Int. J. Biochem. Cell Biol. 35, 606 (2003).
10. H. Paulus, Annu. Rev. Biochem. 69, 447 (2000).
11. P. G. Coulie et al., Proc. Natl. Acad. Sci. U.S.A. 92,
7976 (1995).
12. Y. Guilloux et al., J. Exp. Med. 183, 1173 (1996).
13. R.-F. Wang, M. R. Parkhurst, Y. Kawakami, P. F. Robbins, S. A. Rosenberg, J. Exp. Med. 183, 1131 (1996).
14. J. C. A. Skipper et al., J. Exp. Med. 183, 527 (1996).
15. V. H. Engelhard, A. G. Brickner, A. L. Zarling, Mol.
Immunol. 39, 127 (2002).
16. N. Shastri, S. Schwab, T. Serwold, Annu. Rev. Immunol. 20, 463 (2002).
17. We thank P. Coulie for suggestions, N. Demotte for help
with peptide electroporation, S. Claverol for evaluating the
purity of proteasome preparations, and E. Van Schaftingen,
L. Hue, D. Godelaine, J. Berthet, and E. Warren for critical
reading of the manuscript. N.V. and J.C. were supported by
a Télévie fellowship from the Fonds National de la Recherche Scientifique (FNRS), Belgium. This work was supported
by grants from the FNRS and the Fédération Belge contre
le Cancer (Belgium).
Supporting Online Material
www.sciencemag.org/cgi/content/full/1095522/DC1
Materials and Methods
Fig. S1
References
12 January 2004; accepted 25 February 2004
Published online 4 March 2004;
10.1126/science.1095522
Include this information when citing this paper.
pears to serve functions that are distinct
from those of the T cell receptor (TCR)
coreceptors CD4 and CD8␣␤ (2–4). Immature thymocytes can induce CD8␣␣ upon
strong TCR stimulation (5–7), and in mice
(4, 8–11) and humans (12, 13), CD8␣␣ is
expressed on distinct T cell subsets that
constitutively display a memory phenotype. In light of these characteristics, we
hypothesized that CD8␣␣ might have
functional relevance in specifying T cell
memory fate.
We recently showed that the thymic leukemia antigen TL, a nonclassical major
histocompatibility complex (MHC) class I
molecule, is a unique ligand for CD8␣␣,
with TL tetramers binding specifically to
1
La Jolla Institute for Allergy and Immunology, 10355
Science Center Drive, San Diego, CA 92121, USA.
2
Howard Hughes Medical Institute and Skirball Institute of Biomedical Science, New York University
School of Medicine, New York, NY 10016, USA. 3Department of Pathology and Laboratory Medicine,
Emory University School of Medicine, Atlanta, GA
30322, USA.
*Present address: Institute of Immunology, University
of Vienna, Brunner Strasse 59, 1235 Vienna, Austria.
†To whom correspondence should be addressed. Email: [email protected]
23 APRIL 2004 VOL 304 SCIENCE www.sciencemag.org
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CD8␣␣ but not to CD8␣␤ (4). To determine whether mature T cells induce
CD8␣␣ after TCR stimulation with an antibody to CD3, we stained splenocytes with
TL tetramer. Although no tetramer staining
could be detected on resting splenocytes,
the majority of CD8␣␤⫹ T cells bound TL
tetramer after polyclonal stimulation, and
CD8␣␣ disappeared by day 5 (Fig. 1A).
CD8␣␣ induction was also detected on a
subset of CD8␣␤⫹ OT-1 TCR transgenic
splenocytes stimulated with specific peptide antigen (Fig. 1B). CD8␣␣ expression
was greatest on OT-1 splenocytes stimulated by antigen-presenting cells (APCs) that
had been transfected with TL (APC/TL)
(Fig. 1B); this result suggested that the
interaction of CD8␣␣ with TL might stabilize CD8␣␣ surface expression.
Binding of classical MHC class I tetramers to DP thymocytes and activated T
cells is known to be influenced by modified
glycosylation on CD8␣␤ (14, 15). To exclude the possibility that this accounted for
the observed TL tetramer staining on recently activated CD8␣␤ T cells, we used
chimeric tetramers in which the CD8binding ␣3 domain of TL was replaced by
that of H2-Kb (TL/Kb). Under conditions
where CD8␣␣ was readily detected by TL
tetramers, TL/Kb tetramers failed to stain
activated OT-1 T cells (fig. S1). Addition-
ally, TL tetramer staining of activated OT-1
cells could be blocked by antibodies to
CD8␣ but not by antibodies to CD8␤ (fig.
S1), demonstrating the specificity of the TL
tetramer for CD8␣␣.
Previous studies have suggested that
CD8␣␣ might promote thymocyte survival
(2, 7). We therefore hypothesized that
CD8␣␣ might also rescue mature activated
CD8␣␤ T cells. Consistent with this idea,
CD8␣␣⫹ splenic OT-1 transgenic T cells
that had been activated by APC/TL retained
high levels of the antiapoptotic factors BclxL (Fig. 1C) and Bcl-2 (fig. S2) (16). The
high levels of survival factors were dependent on TL expression by the APC and
could be blocked using antibodies to TL
(Fig. 1C). Accumulation of the antiapoptotic factors also correlated with enhanced
lymphocyte survival, as measured by reduced uptake of Annexin V (Fig. 1C). Activated CD8␣␣⫹ OT-1 TCR splenocytes
also expressed high levels of the shared
interleukin-2 (IL-2)/IL-15 receptor ␤ (R␤)
chain (also called CD122), but not the IL2R␣ chain (CD25) (Fig. 1D). This finding
suggested that these cells expressed the
IL-15 rather than the IL-2 receptor, and this
was supported by the capacity of IL-15 to
expand CD8␣␣⫹ OT-1 CD8⫹ splenocytes,
as compared with an inhibition in the presence of IL-2 (Fig. 1D). Other cytokines,
Fig. 1. CD8␣␣ is transiently induced on activated CD8 splenocytes and provides survival. (A)
C57BL/6 splenocytes cultured
with antibody to CD3␧ were analyzed for CD8␣␣ expression using TL tetramers on activated
(open) and resting (filled)
CD8␤⫹ and CD4⫹ T cells. (B)
CD8⫹ OT-1 TCR splenocytes
cultured with RMAS (APC) or
RMAS-TL (APC/TL) with or without OVA peptide (OVAp) for 3
days were analyzed for CD8␣␣
on gated OVA/H-2Kb tetramer⫹
cells. Data in (A) and (B) are
from one of five independent
experiments. (C) CD8⫹ OT-1
TCR splenocytes cultured with
APC or APC/TL with or without
OVAp for 72 hours. Apoptotic
cells were detected with Bcl-xL
and Annexin-V staining; blocking of Bcl-xL accumulation was
observed in the presence of antibody 18/20 to TL (thin line).
(D) After OVAp stimulation for
72 hours, CD8␣␣⫹ and CD8␣␣⫺
OT-1 TCR splenocytes were analyzed for IL-2R␣ and IL-2/IL15R␤. Data are from one of four
individual experiments. CD8 OT-1 TCR T cells were cultured with the
indicated cytokines and OVAp-loaded irradiated wild-type spleen
cells; the bar graphs show numbers of CD8␣␣⫹ cells on gated V␣2⫹ T
cells (left) and numbers of CD8␣␣⫹ cells and total cells (right). Data
are from one of three individual experiments. (E) Left panels: Total
splenocytes, stimulated as above, were gated on CD11c⫹ or CD11b⫹
including IL-7, had no effect on the expansion of the CD8␣␣⫹ T cells (Fig. 1D).
Costaining of polyclonal activated T cells
with TL tetramers and Annexin V further
demonstrated specific survival of the
CD8␣␣⫹ population (Fig. 1E).
To relate this apparent survival advantage to the interaction of CD8␣␣ with its
ligand, we examined TL expression during
splenocyte stimulation. Although TL was
not detected on resting splenocytes, its expression was observed on activated CD3⫺/
CD11b⫺/c⫹ dendritic cells and CD3⫺/
CD11b⫹/c⫺ monocytes (Fig. 1E), consistent with a potential CD8␣␣/TL-mediated
survival process of the in vitro activated
T cells.
To examine CD8␣␣ induction on T
lymphocytes in vivo, we infected mice
with lymphocytic choriomeningitis virus
(LCMV ) and analyzed them at various
times for expression of CD8␣␣ on LCMVreactive T lymphocytes (16). LCMV
peptide–MHC class I tetramers were used
to identify virus-specific T cells in conjunction with TL tetramers. Induction of
CD8␣␣ was observed on a subset of responding CD8␣␤⫹ LCMV-specific cells by
day 7 after infection (Fig. 2A, day 7). A
proportion of the CD8␣␣⫹ responder cells
did not bind the LCMV-specific tetramers,
most likely because they represented acti-
TCR␤⫺ cells and analyzed for TL expression using antibody HD168 to
TL. Cells were resting (filled), stimulated (open, thick line), or in the presence
of biotinylated antibody to rat isotype (open, thin line). Right panel: Annexin-V and TL tetramer staining of splenocytes stimulated with antibody to
CD3␧ for 72 hours. Data are from one of four independent experiments.
Values in the horizontal bar represent percent positive cells for each staining.
www.sciencemag.org SCIENCE VOL 304 23 APRIL 2004
591
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vated T cells specific for other LCMV
epitopes (Fig. 2A, day 7). The expression
of CD8␣␣ on the LCMV-specific CD8␣␤ T
cells disappeared with virus clearance (Fig.
2A, days 14 and 40) (17). As was the case
in vitro, activated LCMV-specific splenocytes that had induced surface CD8␣␣ expression also expressed high levels of
Bcl-xL (Fig. 2B), consistent with a selective survival advantage for these cells. In
contrast to primary LCMV stimulation
(Fig. 2A, day 7), nearly all the long-term
surviving LCMV-specific CD8⫹ T cells reinduced CD8␣␣ upon secondary stimulation (Fig. 2A, day 60). This is consistent
with the possibility that the capacity to
reinduce CD8␣␣ was selected specifically
during the primary response. The LCMVspecific primary activated CD8␣␣⫹ T cells
also displayed enhanced expression of IL7R␣ (also called CD127) and IL-15R␤
(Fig. 2B), which are typically up-regulated
on memory CD8 T cells (18, 19).
Given the characteristics so far described for CD8␣␣⫹ activated lymphocytes, we reasoned that they might represent precursors of memory T cells. Naı̈ve
CD8⫹ splenocytes isolated from transgenic
mice expressing the P14 TCR (specific for
the LCMV GP33-41 epitope presented by
H-2Db) were transferred into nontransgenic
naı̈ve recipient mice. These animals were
then infected with LCMV and analyzed at
various times after infection. At day 8, a
subset of P14 transgenic donor cells had
induced CD8␣␣, whereas the bulk of primary P14 responder cells remained CD8␣␣
negative (Fig. 3A, day 8). At day 10 after
infection (at the beginning of the contraction phase of the response), the proportion
of CD8␣␣⫹ P14 effector cells remained
unchanged, whereas the CD8␣␣⫺ P14 T
cells decreased markedly (Fig. 3A, day 10).
Similar to wild-type LCMV-specific responder T cells, CD8␣␣⫹ P14 cells also
expressed elevated levels of IL-7R (Fig.
3A). CD8␣␣ expression disappeared by
day 15, however, whereas IL-7R remained
high on the surviving P14 cells (Fig. 3A,
day 15). The selective survival of CD8␣␣expressing primary effector T cells in these
experiments was consistent with their possible role as memory precursors (19).
We next examined the long-term survival of the CD8␣␣⫹ CD8␣␤⫹ T cells and
their potential to mature toward memory T
cells (16). P14 T cells transferred to recipient mice were isolated on day 8 after
LCMV infection and sorted into CD8␣␣⫹
and CD8␣␣⫺ fractions. The two subsets
were then retransferred to naı̈ve recipient
animals. To control for potential migration
differences between the two cell subsets,
we analyzed a set of recipient mice in each
case 2 days after cell transfer for the pres-
592
Fig. 2. CD8␣␣ is selectively induced
on effector CD8 T cells in vivo. (A)
Wild-type mice infected with LCMV
were bled on the indicated days after infection (day 60 mice were rechallenged 3 days before analysis)
and gated CD8⫹ cells were stained
with GP276-286/Db and TL tetramers. (B) Responder cells isolated at
day 7 after infection were analyzed
for intracellular Bcl-xL and surface
expression of IL-2/IL-15R␤ and IL7R␣. Filled and open histograms denote CD8␣␣⫺ and CD8␣␣⫹ effector
cells, respectively. Data are representative of two individual experiments in
(A) and (B) with 30 mice in each experiment. Values shown in each quadrant
of (A) are percentages of tetramer⫹
cells on gated CD8⫹ T cells; percentages
of tetramer⫹ cells of total splenocytes
are in parentheses.
Fig. 3. Direct progeny of
CD8␣␣⫹ effector cells survive and differentiate into
memory T cells. (A) Splenocytes from GP33-41–specific
TCR transgenic mice (P14;
106 cells per mouse) were
transferred into C57BL/6
mice, which were infected
with LCMV 1 day later.
Splenocytes from these animals were analyzed on days
8, 10, and 15 after infection.
After gating on cells stained
for V␣2⫹/V␤8.1⫹, T cells were
analyzed for CD8␣␣ and IL7R␣ expression. Data represent one of four mice per
time point. (B) GP33tet⫹/
CD8␣␣⫹DP and GP33tet⫹SP
sorted primary effector P14
cells (Ly5.2⫹) were transferred a second time into naı̈ve wild-type (Ly5.1) mice (106 cells per mouse). Secondary
recipient mice were rested for 40 days and 3 days after rechallenge with LCMV (⬃105
plaque-forming units per mouse) and analyzed for the recovery and function of Ly5.2⫹
splenocytes. Upper panels: recovered Ly5.2⫹ CD8 cells from one recipient mouse in each case.
Middle panels: IFN-␥ secretion of these cells stimulated with GP33-41 peptide in vitro. Bar
graphs: numbers of recovered P14 cells and IFN-␥–secreting secondary P14 cells from three
recipient mice. Data in (B) are from one of two independent experiments.
ence of the donor cells in various tissues.
Comparable numbers of both cell types
were isolated from each tissue analyzed,
indicating that CD8␣␣⫹ and CD8␣␣⫺ effector cells migrated similarly. Remaining
recipients were rested for 40 days and analyzed after rechallenge with the virus.
Substantial numbers of P14 T cells were
recovered from mice that had received
CD8␣␣⫹ primary P14 effector cells, but
not from those that had received the
CD8␣␣⫺ fraction (Fig. 3B). To test for
functional differentiation of the surviving
donor cells, we measured cytokine responses after in vitro stimulation with GP33-41
peptide. Most progeny of CD8␣␣⫹ P14
effector cells displayed a potent secondary
response, as measured by intracellular interferon-␥ (IFN-␥) staining, whereas
CD8␣␣⫺ progeny did not (Fig. 3B).
As a direct test for a role of CD8␣␣ in
memory formation, mice carrying an enhancer deletion within the murine cd8 locus
(E8I⫺/⫺) (20) were examined. This enhancer is required for expression of CD8␣␣, and
although cells from these mice express normal levels of CD8␣␤ (20), no CD8␣␣
could be detected after stimulation of
E8I⫺/⫺ T cells in vitro (fig. S3). These
observations indicated that the induction of
CD8␣␣ on activated CD8␣␤⫹ splenocytes
is controlled by the E8I enhancer. Consis-
23 APRIL 2004 VOL 304 SCIENCE www.sciencemag.org
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Fig. 4. CD8␣ enhancer– deficient
mice (E8I⫺/⫺) are compromised in
generating memory CD8 T cells.
Wild-type (WT) and E8I⫺/⫺ mice infected with LCMV were analyzed for
primary, memory, and secondary response on days 7, 50, and 56 after
rechallenge, respectively. (A) Gated
NP396 tet⫹, GP276 tet⫹, or GP33
tet⫹ CD8 primary effector T cells analyzed for CD8␣␣. Data are from one
of four mice in each group. (B)
Splenocytes stained for GP33/Db tetramer on gated CD8⫹ cells were assessed for LCMV-specific primary and
secondary responses. (C) Dot plots of
IFN-␥ production from primary,
memory, and secondary responder
cells measured after in vitro restimulation with GP33-41 peptide. Values
indicate percentage of IFN-␥⫹ cells in
total splenocytes. Bar graphs show
percentages of IFN-␥ ⫹ primary,
memory, or secondary LCMV effector
cells from WT or E8I⫺/⫺ mice and
stimulated in vitro with GP33-41 or
NP396-404 peptide. Data in (B) and
(C) are from 9 to 12 mice each. (D) LCMV-specific primary effector cells gated on GP33/Db tetramer⫹ cells were stained for CD8␣␣ and IL-7R␣ expression.
Data are from one of five mice.
tent with this idea, LCMV-infected E8I⫺/⫺
mice generated substantial numbers of primary LCMV-specific CD8␣␤⫹ effector
cells (Fig. 4, A and B), none of which
expressed CD8␣␣ (Fig. 4A). Analysis of
E8I⫺/⫺ and wild-type mice for primary responses revealed comparable amounts of
IFN-␥–producing primary T cells from
both mice (Fig. 4C). In contrast, the low
numbers of E8I⫺/⫺ LCMV-specific T cells
isolated 50 days after the initial infection,
and the poor response of these cells upon
restimulation in vitro, indicated a severe
defect of the E8I⫺/⫺ lymphocytes in terms
of survival and memory cell differentiation
(Fig. 4, B and C). Additionally, IL-7Rhigh
cells were also absent among primary
E8I⫺/⫺ effector cells, indicating that
CD8␣␣ is required for the initial upregulation of IL-7R on CD8 memory T cell
precursors (Fig. 4D).
Unique features of CD8␣␣ distinguish
this molecule from the conventional TCR
coreceptor CD8␣␤. CD8␤ facilitates translocation of the CD8 coreceptor into lipid rafts
and localization of the CD8␣-associated
p56lck in proximity to the TCR activation
complex (21). CD8␣␣ is largely excluded
from these rafts (21) and could thus sequester
p56lck away from the TCR. Dual expression
of CD8␣␣ with CD8␣␤ also appears to mediate raft disruption and an overall decrease
of TCR activation signals (22). However, the
effect of CD8␣␣ may not simply be inhibitory, as CD8␣␣ might redirect the p56lck
toward other immune receptors outside rafts,
including IL-7R and IL-15R.
The impaired CD8 T cell memory observed in E8I⫺/⫺ mice provides direct evi-
dence that CD8␣␣ is a key player in the
initial survival of primary effector cells as
well as ultimately in memory differentiation
itself. The activation-induced expression of
TL on APCs at the same time as CD8␣␣ on
T cells suggests that CD8␣␣ is likely to
mediate its function through interaction with
TL. TL is not a typical peptide-binding MHC
molecule (23), and the preferential interaction of TL with CD8␣␣ (4) could potentially
have the effect of further reinforcing the sequestration of p56lck away from the TCR.
Although at this stage the CD8␣␣dependent mechanism for memory formation
described here appears to be confined to
CD8␣␤⫹ T cells, the previous observations
that under certain activation conditions CD4
T cells can induce CD8␣␣ (24, 25) leave
open the possibility that a similar process
might also operate in CD4 T cell memory.
Human CD8␣␤⫹ TCR␣␤⫹ peripheral blood
lymphocytes also express CD8␣␣, and human CD8␣␣⫹ T cells with a memory phenotype have recently been identified (13), suggesting that CD8␣␣-dependent memory formation may be preserved across species.
In the design of vaccines, much attention
has been focused on the magnitude of the
initial immune effector cell burst in terms of
establishing robust immune memory (26).
Our results indicate that this is not the sole
determining factor and that selective survival
and differentiation of CD8␣␣⫹ precursors,
leading to long-lived CD8 memory T cells,
also plays an important role.
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27. We thank M. Cheroutre and O. Turovskaya for their
contributions; M. Kronenberg, J. Braun, E. Reinherz
and E. Sercarz for helpful discussions; and C. Miceli, J.
Altman, R. Ahmed, N. Shastri, and W. Heath for
constructs, reagents, cell lines, and mice. Supported
by NIH grants DK54451 and AI50263 (H.C., L.T.M.),
AI33614 (P.J.), and AI51973 (M.G.V.) and by Juvenile
Diabetes Research Foundation grant 3-2000-510
(U.C.). This is manuscript number 512 of the La Jolla
Institute for Allergy and Immunology.
Supporting Online Material
www.sciencemag.org/cgi/content/full/304/5670/590/DC1
Materials and Methods
Figs. S1 to S3
References
7 October 2003; accepted 18 February 2004
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