Cutting Edge: Memory CD8 T Cell
Maturation Occurs Independently of CD8 αα
Anmol Chandele and Susan M. Kaech
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References
J Immunol 2005; 175:5619-5623; ;
doi: 10.4049/jimmunol.175.9.5619
http://www.jimmunol.org/content/175/9/5619
OF
THE
JOURNAL IMMUNOLOGY
CUTTING EDGE
Cutting Edge: Memory CD8 T Cell Maturation Occurs
Independently of CD8␣␣1
Anmol Chandele and Susan M. Kaech2
M
emory T cells are critical for mediating long-term
protective immunity against infectious disease, but
only a few of the key signals and pathways that govern their formation have been elucidated. Recent work has
shown that IL-7 is essential for generation and maintenance of
both memory CD8 and CD4 T cell populations (Ref. 1 and
references within). The ability of an effector CD8 T cell to survive and become a memory CD8 T cell relies on both the availability of IL-7, as well as the effector cell’s ability to express the
IL-7R ␣-chain (IL-7R␣)3 (2– 4). During an acute viral or bacterial infection, the expression of IL-7R␣ is down-regulated in
the majority of activated effector CD8 T cells, but at the peak of
the CD8 T cell response, a small subset of effector CD8 T cells
express higher levels of IL-7R␣ (and these are referred to as IL7R␣high cells) (2–5). Additional experiments showed that the
IL-7R␣high effector CD8 T cells preferentially survive and become the long-lived memory CD8 T cells that protect against
reinfection, making IL-7R␣ a marker that distinguishes the acSection of Immunobiology, Yale University School of Medicine, New Haven, CT 06520
Received for publication July 8, 2005. Accepted for publication September 2, 2005.
The costs of publication of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
tivated T cells that will survive (i.e., memory cell precursors)
from those that will die following infection (2, 4).
A molecule recently implicated in directing IL-7R␣ expression on memory CD8 T cell precursors is the homodimer
CD8␣␣ (5). CD8␣␣ is a ligand for the nonclassical MHC molecule thymic leukemia (TL) Ag and can be detected with TL
tetramers (6). One study found that CD8␣␣ was coexpressed
transiently on IL-7R␣high effector CD8 T cells during lymphocytic choriomeningitis virus (LCMV) infection (between days 7
and 14 postinfection (p.i.)). Moreover, IL-7R␣high effector
CD8 T cells were not detected at the peak of clonal expansion
(day 7 p.i.) in animals that are significantly defective in expressing CD8␣␣ homodimers (5). These mutant animals (referred
to as E8I⫺/⫺ mice) contain a deletion of the E8I enhancer in the
intergenic region of the CD8 ␣␤ gene complex (7). Formation
of protective memory CD8 T cells was also greatly impaired in
E8I⫺/⫺ mice, presumably due to defective IL-7R␣high memory
CD8 T cell precursor development (5).
Several studies have highlighted that functionally competent
memory CD8 T cells form over several weeks to months following acute infection, and during this maturation period, several changes are observed in gene expression and functional responses (Ref. 8 and references within). When integrated, these
observations suggest that CD8␣␣ acts early to induce formation of IL-7R␣high memory CD8 T cell precursors, but their
subsequent maturation into memory CD8 T cells is conducted
independently of CD8␣␣. Because of this temporal discordance between CD8␣␣ expression and memory CD8 T cell
maturation, we aimed to determine whether CD8␣␣ controlled other aspects of memory CD8 T cell development, in
addition to IL-7R␣ expression. Through a comprehensive analysis of effector and memory CD8 T cell differentiation during
LCMV infection, we found that the generation of memory
CD8 T cells, based on number, form, and function, was relatively normal in E8I⫺/⫺ mice compared with wild type (WT;
C57BL/6). Our data indicate that CD8␣␣ is not a primary signal controlling IL-7R␣ expression on memory CD8 T cell precursors or their development into long-lived memory CD8 T
cells that can protect against secondary infection.
2
Address correspondence and reprint requests to Dr. Susan M. Kaech, 300 Cedar Street,
TAC S641B, Yale University School of Medicine, P.O. Box 208011, New Haven, CT
06520. E-mail address: [email protected]
Abbreviations used in this paper: IL-7R␣, IL-7R ␣-chain; TL, thymic leukemia; LCMV,
lymphocytic choriomeningitis virus; p.i., postinfection; WT, wild type; IEL, intraepithelial
lymphocyte; CD62L, L-selectin.
3
1
This work was supported by Burroughs-Wellcome Fund 1004313 (to S.M.K.), National
Institutes of Health Grant R01 AI 066232-01 (to S.M.K.), Edward Mallinckrodt, Jr.,
Foundation (to S.M.K.), and Cancer Research Institute (to S.M.K.).
Copyright © 2005 by The American Association of Immunologists, Inc.
0022-1767/05/$02.00
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
As memory CD8 T cells form during acute viral infection,
several changes in gene expression and function occur, but
little is known about the control of this process. It was reported previously that the homodimer CD8␣␣ was involved
in generating IL-7R␣high memory CD8 T cell precursors,
and consequently, protective memory CD8 T cells did not
form in animals significantly impaired in CD8␣␣ expression (E8Iⴚ/ⴚ mice). However, the precise contribution of
CD8␣␣ to sustained IL-7R␣ expression and other memory
CD8 T cell-associated changes has not been investigated. We
found that IL-7R␣ expression and generation of memory
CD8 T cells that protect against secondary viral infection was
considerably normal in E8Iⴚ/ⴚ animals. Interestingly, virusspecific CD4 T cell responses were elevated, and the relative
surface levels of CD8␣␤ in activated T cells were reduced in
E8Iⴚ/ⴚ mice compared with wild-type animals. Our results
indicate that memory CD8 T cell development can occur independently of CD8␣␣. The Journal of Immunology,
2005, 175: 5619 –5623.
5620
CUTTING EDGE: MEMORY CD8 T CELL DEVELOPMENT IN E8I-DEFICIENT ANIMALS
Materials and Methods
LCMV-specific IgG ELISA
Mice and viral infection
LCMV-specific IgG Ab titers in sera were determined by solid-phase ELISA, as
described previously (9).
C57BL/6J (WT) mice were purchased from The Jackson laboratory, and
E8I⫺/⫺ knockout mice were kindly provided by D. Littman (Skirball Institute,
New York, NY) and H. Cheroutre (La Jolla Institute for Allergy and Immunology, San Diego, CA). Mice were infected with 2 ⫻ 105 PFU of LCMVArmstrong i.p. or 2 ⫻ 106 PFU of LCMV-clone 13 i.v. Viral titers were quantified from serum and tissues of infected mice by plaque assay on Vero cell
cultures as described previously (9). The animals were housed and used under
approved institutional animal care and use committee protocols.
Genotyping of E8I⫺/⫺ mice
Genomic DNA was extracted from blood by DNeasy tissue kit (Qiagen) according to the manufacturer’s instructions and PCR amplified with gene-specific primers for WT and E8I alleles: a common forward primer, 5⬘-ATTC
CCAACACCCACTACAAG-3⬘, reverse WT primer, 5⬘-AGCTATCTTCA
GACGTGTCAG-3⬘, and reverse E8I primer, 5⬘-GGGGCTATAGCTCTG
TAGGTCA-3⬘ that amplifies a 1.3- and a 1.6-kb product, respectively. Annealing temperatures for the WT and mutant primer sets was 54°C and 57°C,
respectively.
Lymphocyte isolation and cell surface and intracellular cytokine staining
Normal IL-7R␣ expression by LCMV-specific effector and memory CD8
T cells in E8I-deficient mice
Before initiating our studies, the genotype of the E8I⫺/⫺ mice
was verified by using functional and molecular assays (data not
shown). By staining IELs for CD3, CD8␣, CD8␤, and TL tetramers, we confirmed that the development of CD8␣␣ IELs
was greatly reduced in the E8I⫺/⫺ mice as shown previously (5,
7). Their genotype was also verified by PCR, which showed that
all E8I⫺/⫺ mice in our studies are homozygous for this deletion.
To examine the expression of IL-7R␣ on effector and memory CD8 T cells, we infected WT and E8I⫺/⫺ mice with
LCMV and 8, 15, and 45 days later, cells were isolated from the
blood and spleen and stained with Abs to CD8␣ and IL-7R␣
and MHC class I tetramers DbGP33– 41 and DbNP396 – 404,
which bind LCMV-specific CD8 T cells (Fig. 1A and data not
shown). At day 8 p.i., ⬃5–15% of LCMV-specific effector
CD8 T cells expressed IL-7R␣ in both WT and E8I⫺/⫺ mice
(Fig. 1A). Over the next several weeks, the proportion of IL7R␣high LCMV-specific CD8 T cells in WT and E8I⫺/⫺ mice
continued to increase in an identical pattern. By day 45 p.i.,
between 70 and 80% of the memory CD8 T cells in both WT
and E8I⫺/⫺ mice expressed high amounts of IL-7R␣ (Fig. 1A).
FIGURE 1. Normal IL-7R␣ expression pattern and numbers of LCMV-specific CD8 T cells in E8I-deficient animals. A, WT and E8I⫺/⫺ mice were infected with
LCMV-Armstrong, and on days 8, 15, and 30 –55 p.i., the lymphocytes were isolated from the spleen, liver, and blood and stained for CD8␣, IL-7R␣, and
DbGP33– 41 MHC class I tetramers. Plots on left show percentage of CD8 T cells (bottom numbers) and of tetramer⫹ CD8 T cells (top numbers), and plots on the right
show percentage of IL-7R␣high GP33– 41-specific CD8 T cells. The frequency of NP396 – 404- and GP276 – 86-specific CD8 T cells and their expression of IL-7R␣ was
also similar between WT and E8I⫺/⫺ mice (data not shown). B, Bar graph shows the combined numbers of LCMV-specific CD8 T cell populations (NP396 – 404,
GP33– 41/34 – 41, GP276 – 86, and NP205–212) in the spleen that were calculated based on intracellular IFN-␥ staining after 5 h of stimulation with peptides. C, Splenocytes from WT and E8I ⫺/⫺ mice (⬃30 –55 days p.i.) were stimulated with NP396 – 404 and GP33– 41/34 – 41 peptides for 5 h and stained for CD8␣, IFN-␥, and IL-2.
Left panel shows the percentage of IFN-␥⫹ CD8 T cells, and the right panel (gated on IFN-␥⫹ cells) shows the percentage of cells that coproduce IL-2. D, Splenocytes
from WT and E8I⫺/⫺ mice infected ⬃50 days prior were stained for CD8␣, CD62L, CD27, and DbGP33– 41 MHC class I tetramers. Histograms show CD62L and
CD27 expression levels on tetramer⫹ CD8 T cells.
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Lymphocytes were isolated from spleen, blood, liver, lung, and intestine as described previously (10). Intraepithelial lymphocytes (IELs) were isolated from
the intestine as described in Ref. 11. Abs against IL-7R␣ (CD127), CD3,
CD8␣, B220, IFN-␥, IL-2, and TNF-␣ were purchased from eBioscience, and
CD4, Fas, and IgD were purchased from BD Biosciences. H-2Db tetramers
bound to LCMV peptides NP396 – 404, GP276 – 86, and GP33– 41 were generated
as described previously (12). Surface and intracellular cytokine staining was performed as described previously (12). Cells were stained with TL tetramer (2.5–5
␮g/ml), and Abs to CD3, CD4, and CD44 or for 30 min on ice (and CD8 T
cells were examined by gating on CD3⫹CD4⫺ cells), or TL tetramer was added
first followed by the addition of Abs to CD8␣, CD8␤, and CD44.
Results and Discussion
The Journal of Immunology
5621
Memory CD8 T cell maturation appears normal in E8I-deficient
animals
Because the LCMV-specific CD8 T cells that survived the contraction phase in E8I⫺/⫺ mice showed a typical pattern of IL7R␣ expression, we next examined the numbers of memory
CD8 T cells that formed. The frequency and number of
LCMV-specific CD8 T cells in the spleens of E8I⫺/⫺ and WT
mice were calculated at days 8, 15, and 30 –55 p.i. (Fig. 1).
Using both MHC class I tetramer staining (Fig. 1A) and
intracellular cytokine staining for IFN-␥ (Fig. 1C), we found
similar numbers of CD8 T cells specific for the dominant
(DbNP396 – 404, DbGP33– 41, and KbGP34 – 41) and subdominant (DbGP276 – 86 and DbNP205–12) LCMV epitopes in WT
and E8I⫺/⫺ mice at all time points (Fig. 1B). At days 8 and
15 p.i., both groups of mice contained ⬃20 –25 ⫻ 106 and
3–5 ⫻ 106 LCMV-specific CD8 T cells, respectively. The
similarity in numbers at day 15 p.i. indicates that effector cell
apoptosis was not occurring at higher than normal rates in
E8I⫺/⫺ mice. At days 30 –55 p.i., the WT and E8I⫺/⫺ mice
contained equivalent numbers of LCMV-specific memory
CD8 T cells ranging from ⬃1 to 3 ⫻ 106 cells/spleen (Fig. 1, B
and C). Similar numbers of memory CD8 T cells were also
found in the liver, lung, and blood of E8I⫺/⫺ mice, indicating
that memory CD8 T cells were also generated in nonlymphoid
organs (Fig. 1A and data not shown).
Importantly, the maturation of memory CD8 T cells also appeared to occur normally in E8I⫺/⫺ mice based on expression of
IL-7R␣, L-selectin (CD62L), CD27, and production of IL-2
(Fig. 1, C and D). At days 30 –55 p.i., the memory CD8 T cells
in both WT and E8I⫺/⫺ mice were ⬃70 – 80% IL-7R␣high,
⬃20 –30% CD62Lhigh, ⬃40 –50% CD27high, and ⬃15–25%
could produce IL-2 (13). Taken together, it appeared that
memory CD8 T cell differentiation and survival was not defective in animals lacking CD8␣␣ expression.
Increased virus-specific CD4 T cell responses in E8I-deficient animals
In addition to the CD8 T cell responses, the virus-specific CD4
T and B cell responses were examined. The number of LCMVspecific CD4 T cells specific for the GP61– 80 epitope was measured at days 8 and 45 p.i. by intracellular cytokine staining for
IFN-␥ and TNF-␣ (Fig. 2A). Interestingly, there was a 2- to
FIGURE 2. CD4 T cell and B cell responses in E8I⫺/⫺ mice during LCMV
infection. A, Splenocytes from WT and E8I⫺/⫺ mice infected 8 and 45 days
previously were stimulated with GP61– 80 peptide for 5 h and stained for CD4,
IFN-␥, and TNF-␣. Plots are gated on CD4 T cells and show the percentage of
IFN-␥⫹ and TNF-␣⫹ cells. Bar graph on right shows numbers of GP61– 80specific CD4 T cells in the spleen in WT (f) and E8I⫺/⫺ (䡺) animals. B,
LCMV-specific IgG were measured in the serum of uninfected animals (naive)
or in animals infected ⬃40 –55 days previously using ELISA.
4-fold increase in the number effector and memory GP61– 80specific CD4 T cells in E8I⫺/⫺ mice. This correlated with a
slight increase in the number of germinal center B cells in
E8I⫺/⫺ mice at day 15 p.i. as assessed by flow cytometry of
B220⫹, IgD⫺, PNA⫹, Fas⫹ B cells, but there was no apparent
difference in the titers of LCMV-specific IgG Abs compared
with WT mice (Fig. 2B and data not shown). Although the underlying cause for the elevated CD4 T cell response is not clear,
it is possible that CD8␣␣ functions in another cell type, such as
CD8␣⫹ dendritic cells, to negatively regulate CD4 T cell expansion. Further investigation of this finding is needed.
The E8I enhancer is needed for optimal CD8␣ expression in activated
CD8 T cells
During the above experiments, we noted that amounts of surface CD8␣ was lower in a portion of the activated LCMV-specific CD8 T cells in E8I⫺/⫺ mice when analyzed directly ex vivo
or after 5 h of stimulation in vitro (Figs. 1C, 3, and 4C). At day
8 p.i. the CD8␣ median fluorescent intensity was ⬃20 – 45%
lower in E8I⫺/⫺ animals compared with WT (Fig. 3). Because
murine CD8␤ cannot traffic to the plasma membrane independent of CD8␣ (14), CD8␤ was decreased correspondingly. As
time postinfection proceeded, the reduced CD8␣␤ expression
became less apparent (days 15 and 45; Fig. 3). However, the
CD8␣ down-regulation was observed again 5 and 8 days after
secondary LCMV-cl.13 infection (Figs. 3 and 4C and data not
shown). The extent of CD8␣ down-regulation was variable between E8I⫺/⫺ mice, but it was consistently observed in over 30
animals analyzed. Thus, it appears that the E8I enhancer may
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The frequency of NP396 – 404- and GP276 – 86-specific CD8 T
cells and their expression of IL-7R␣ was also similar between
WT and E8I⫺/⫺ mice (data not shown). These data show that
neither the initial formation of IL-7R␣high memory CD8 T cell
precursors nor their ability to maintain IL-7R␣ expression as
they matured into memory CD8 T cells was abrogated by the
lack of CD8␣␣ during acute viral infection.
The expression of CD8␣␣ on effector CD8 T cells at day
8 p.i. was also examined to see if there was a correlation between
IL-7R␣ and CD8␣␣ expression as reported previously (5). Using TL tetramers, we failed to reproducibly identify a clear population of CD8␣␣⫹ effector CD8 T cells in the spleens of WT
animals, whereas the same staining protocols routinely detected
CD8␣␣⫹ T cells within the IEL population, indicating that the
TL tetramer was functional (data not shown). Perhaps more optimized staining protocols than those used here are required to
detect CD8␣␣⫹ effector CD8 T cells in the spleen because
their overall amounts of CD8␣␣ expression is significantly
lower than that of IELs.
5622
CUTTING EDGE: MEMORY CD8 T CELL DEVELOPMENT IN E8I-DEFICIENT ANIMALS
Protective memory CD8 T cell recall responses in E8I-deficient mice
FIGURE 4. Protective memory CD8 T cell responses in E8I ⫺/⫺ mice. A–C,
WT and E8I⫺/⫺ mice were infected with LCMV-Armstrong, and ⬃50 –55
days later, the mice were reinfected with LCMV-cl.13 and analyzed 8 days later.
A, Bar graph shows cl.13 virus titers in the spleen at day 8 p.i. Naive mice were
infected for a positive control of infection. Dashed line denotes level of detection. B, Bar graph shows the combined numbers of DbNP396 – 404, DbGP33– 41,
and DbGP276 – 86-specific CD8 T cells in the spleen 8 days p.i. that were calculated based on MHC class I tetramer staining. C, IL-7R␣ expression on NP396 –
404- and GP33– 41-specific CD8 T cells 8 days p.i. Left panels show percentage of
CD8 T cells (bottom numbers) and tetramer⫹ CD8 T cells (top numbers). Right
panels show percentage of IL-7R␣hightetramer⫹ CD8 T cells. Note the reduction in CD8␣ expression on tetramer⫹ CD8 T cells in E8I⫺/⫺ mice; these
CD8␣low cells were not included in the calculations shown in B.
The above data showed that the formation of memory CD8 T
cells was not defective in E8I⫺/⫺ mice. Therefore, we tested the
ability of the memory CD8 T cells to protect against a secondary LCMV infection by reinfecting LCMV-immune WT and
E8I⫺/⫺ mice with a more virulent strain of LCMV (cl.13). WT
immune animals mount rapid recall responses and clear the virus within 5 days (9). No virus was detected in the serum (day
5 p.i., data not shown) or in the spleen (day 8 p.i.) in either WT
or E8I⫺/⫺ mice, indicating that the E8I⫺/⫺ mice can efficiently
clear a secondary LCMV-cl.13 infection (Fig. 4A). Overall, the
secondary clonal expansion of the memory CD8 T cells was
very similar between WT and E8I⫺/⫺ mice, and both groups
contained ⬃8 –10 ⫻ 106 LCMV-specific CD8 T cells at day
8 p.i. (Fig. 4B). When the different epitope-specific CD8 T cell
populations were analyzed individually, small differences between WT and E8I⫺/⫺ mice were observed. WT mice contained ⬃25–30% more DbNP396 – 404- and DbGP276 – 86-specific CD8 T cells in their spleens at day 8 p.i. compared with
E8I⫺/⫺ mice (Fig. 4C and data not shown). However, there was
a compensatory ⬃30% increase in the number of DbGP33– 41specific CD8 T cells in the E8I⫺/⫺ compared with WT mice.
Thus, the total number of LCMV-specific secondary effector
CD8 T cells was very similar between WT and E8I⫺/⫺ mice.
The pattern of IL-7R␣ expression on these cells did not differ
between the two groups, again indicating that virus-specific
CD8 T cells can express IL-7R␣ independent of CD8␣␣ signals (Fig. 4C). Altogether, these data show that the LCMV-specific memory CD8 T cells in E8I⫺/⫺ mice were capable of profound expansion and viral clearance in response to a secondary
infection, indicating that their protective responses were intact.
In summary, our analysis of the E8I⫺/⫺ mice indicates that
CD8␣ signals are not necessary for IL-7R␣ expression or for
memory CD8 T cell formation during LCMV infection. However, it should be noted that the expression of CD8␣␣ in
E8I⫺/⫺ mice is significantly reduced but not entirely ablated
(7). Therefore, it is plausible that greatly reduced to undetectable amounts of CD8␣␣ are sufficient to promote memory
CD8 T cells to develop. A better test of this model may be to
develop transgenic animals that contain specific mutations in
CD8␣ that inhibit CD8␣␣ homodimer but not CD8␣␤ heterodimer formation. The results of such a study should be
revealing.
The memory CD8 T cells in E8I-deficient animals could
mount protective recall responses and control secondary viral
infection. The overall secondary expansion of memory CD8 T
cells in E8I⫺/⫺ mice following LCMV reinfection was similar to
that of WT mice; however, minor differences were noted when
different epitope-specific CD8 T cell populations were examined. The reduction in CD8␣␤ expression was acutely evident
on the secondary effector CD8 T cells during LCMV-cl.13 reinfection in E8I⫺/⫺ animals, and perhaps the decreased expression of CD8␣␤ contributed to the reduced expansion or survival of NP396 – 404- and GP276 – 86- specific effector CD8 T
cells. Recent work shows that lowered amounts of surface
CD8␣ can exacerbate proliferative and functional CD8 T cell
responses (15). Therefore, it is a formal possibility that variations in CD8 T cell responses in the E8I⫺/⫺ mice can be attributed to reduced levels of CD8␣␤ rather than, or in addition, to
CD8␣␣.
An underlying aspect of many models of memory CD8 T cell
development is that the “strength of signal” experienced by a T
cell impacts its ability to become a memory CD8 T cell (16, 17).
operate to maintain normal expression of CD8␣␤ on recently
activated CD8 T cells.
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FIGURE 3. Decreased expression of CD8␣␤ is observed in activated CD8
T cells in E8I-deficient mice. Splenocytes from WT and E8I⫺/⫺ mice infected
8, 15, and 45 days previously with LCMV-Armstrong were stained for CD8␣,
CD8␤, and CD44. Bottom row shows data from LCMV immune animals 8
days after LCMV-cl.13 reinfection. The median fluorescent intensity of CD8␣
and CD8␤ is indicated in lower right quadrant of left panels. Arrows highlight
CD44high CD8 T cells with reduced CD8␣ expression.
The Journal of Immunology
Acknowledgments
We thank T. Pasqualini for technical assistance, Drs. G. Shadel, E. J. Wherry,
M. Krishna, R. Ahmed, F. Lakkis, G. Chalasani, and J. Obhrai and N. Joshi for
critical reading of the manuscript, and W. Ellmeier for PCR protocols.
H. Cheroutre kindly provided the TL-tetramer and E8I⫺/⫺ mice (with permission from D. Littman).
Disclosures
The authors have no financial conflict of interest.
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Indeed, the proposed role of CD8␣␣ in memory CD8 T cell
development is to down-modulate TCR signals and promote
effector cell survival by sequestering lck away from the TCR via
its exclusion from lipid rafts (5). This model holds much credibility, and our studies here do not directly examine this point.
Our results indicate that if TCR signaling is heightened in
E8I⫺/⫺ animals (as put forth in Ref. 5), then this level is not
sufficient to inhibit formation of IL-7R␣high memory CD8 T
cell precursors or their ability to develop into protective memory CD8 T cells during acute LCMV infection. The reason(s)
for the discrepancies between our study and Madakamutil et al.
(5) is not clear because, for the primary infection, similar doses
of LCMV-Armstrong and routes of infection were used. Therefore, the generation of IL-7R␣-expressing memory CD8 T cells
in E8I⫺/⫺ mice cannot be attributed to differences in viral infection. Moreover, a recent study by Zhong and Reinherz (18)
have found that the formation of functional memory CD8 T
cells also occurs normally in E8I⫺/⫺ mice after influenza infection. Another recent study by Williams and Bevan have demonstrated that a single MHC class Ia molecule is sufficient for
formation of memory CD8 T cells, suggesting that TL signals
are not required for memory CD8 T cell development (19). Altogether, these studies question the role of CD8␣␣ in memory
CD8 T cell differentiation, and thus, more direct and comprehensive studies are needed.
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