Activated CD8 T Cells Redistribute to
Antigen-Free Lymph Nodes and Exhibit
Effector and Memory Characteristics
This information is current as
of June 16, 2017.
C. Colin Brinkman, Stacey L. Sheasley-O'Neill, Andrew R.
Ferguson and Victor H. Engelhard
J Immunol 2008; 181:1814-1824; ;
doi: 10.4049/jimmunol.181.3.1814
http://www.jimmunol.org/content/181/3/1814
Subscription
Permissions
Email Alerts
This article cites 55 articles, 23 of which you can access for free at:
http://www.jimmunol.org/content/181/3/1814.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2008 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
References
The Journal of Immunology
Activated CD8 T Cells Redistribute to Antigen-Free Lymph
Nodes and Exhibit Effector and Memory Characteristics1
C. Colin Brinkman,2 Stacey L. Sheasley-O’Neill,2,3 Andrew R. Ferguson,
and Victor H. Engelhard4
O
ver the last several years, much has been done to elucidate the processes of CD8 T cell activation and differentiation in vivo. The interactions between Ag-specific
T cells and Ag-presenting dendritic cells (DC)5 during the early
stages of T cell activation have been nicely detailed using twophoton microscopy (1–3). Similarly, tracking the entry of soluble
Ag into individual lymph nodes (LN) after s.c. injection has provided insight into multiple stages of Ag presentation by LN-resident and skin-derived DC (4). In addition, the distribution of activated effectors into peripheral nonlymphoid tissues at later times
during the primary response has also been evaluated in detail (5–
9). However, the understanding of intermediate stages of T cell
differentiation, and their progression within individual lymphoid
compartments is incomplete. Many previous studies have used
pathogens that activate systemic responses, and the ensuing T cell
responses have been examined only in the spleen (10 –14). Because the spleen is both a T cell activation site and a repository for
effectors that may have been activated in other sites, these analyses
do not allow a clear understanding of the relationship of the site of
Department of Microbiology and Carter Immunology Center, University of Virginia,
Charlottesville, VA 22908
Received for publication January 28, 2008. Accepted for publication May 28, 2008.
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.
1
This work was supported by United States Public Health Service Grant CA78400 (to
V.H.E.), United States Public Health Service Training Grants GM07627 (to S.S.O.),
AI007496 and the Robert R. Wagner Fellowship (to C.C.B.), and United States Public
Health Service fellowship AI072818 (to A.R.F.).
2
These authors contributed equally to this work and their order should be considered
arbitrary.
3
Current Address: Department of Pathology and Laboratory Medicine, University of
North Carolina, Chapel Hill, North Carolina 27599.
4
Address correspondence and reprint requests to Dr. Victor H. Engelhard, Carter
Immunology Center, University of Virginia, Box 801386, Charlottesville, VA 229081386. E-mail address: [email protected]
5
Abbreviations used in this paper: DC, dendritic cell; LN, lymph nodes; BMDC,
bone marrow-derived dendritic cell; TCM, central T memory.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
www.jimmunol.org
CD8 T cell activation to subsequent differentiation and migration
patterns. Several other studies have used routes of viral infection
that resulted in initially localized Ag presentation in LN and examined CD8 T cells exclusively in draining LN or spleen (6, 15–
18). A few of these studies have shown that activated CD8 T cells
are present in both draining and presumptive nondraining LN (19,
20). However, these studies reached conflicting conclusions about
whether activated cells in presumptive nondraining nodes have
migrated there, or been activated there at a later stage in the response due to dissemination of the virus (15, 21, 22).
A second area of uncertainty, intertwined with the first, is the
relationship of effector cell differentiation to that of memory cells.
The differentiation of a stable memory population after viral infection has been reported to require at least 40 days (23). However,
it has been argued that this is an artifact of using a large number
of adoptively transferred T cells and that memory populations are
established much sooner (24). In keeping with this, memory cell
precursors have been identified in draining LN as early as 3.5 days
after response initiation, based on their survival after adoptive
transfer (18), and as early as 7 days based on re-expression of
IL-7R␣ (CD127) (25). It has also recently been suggested that
memory and effector lineages are produced by asymmetric cell
division that is evident in the first daughter cells arising from a
newly activated T cell (26). These observations highlight uncertainty over whether memory T cells differentiate as a distinct
population (27, 28), or represent early stage effectors programmed to survive (24, 25, 29, 30). This issue has been further
complicated by the identification of memory cell subsets (6,
31). Central memory (TCM) cells are localized to LN, and are
generally considered to express CCR7 and high levels of
CD62L. In contrast, effector memory cells are localized to peripheral tissue and are generally considered negative for these
two markers. Different studies suggest either that one of these
populations gives rise to the other (24, 25, 29, 30) or that they
arise independently (27, 28). These models also have not addressed the question of whether precursors of TCM cells exit and
then recirculate among LN, or complete their differentiation in
the LN in which they were initially activated.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Exogenous dendritic cells display restricted trafficking when injected in vivo and stimulate CD8 T cell responses that are localized
to a small number of lymphoid compartments. By examining these responses in the presence and absence of FTY720, a drug that
causes sequestration of T cells in lymph nodes, we demonstrate that a significant fraction of divided CD8 T cells redistribute into
Ag-free lymph nodes within 3 days of activation. Despite variation in the level of expression of CD62L, redistribution of these cells
is CD62L-dependent. Redistributed CD8 T cells exhibit characteristics of differentiated effectors. However, when re-isolated from
Ag-free lymph nodes 3 days after activation and transferred into naive mice, they persist for at least 3 wk and expand upon Ag
challenge. Thus, CD8 T cells that redistribute to Ag-free lymph nodes 3 days after immunization contain memory precursors. We
suggest that this redistribution process represents an important mechanism for establishment of lymph node resident central
memory, and that redistribution to Ag-free nodes is an additional characteristic to be added to those that distinguish memory
precursors from terminal effectors. The Journal of Immunology, 2008, 181: 1814 –1824.
The Journal of Immunology
Materials and Methods
Mice, viruses, and viral infection
C57BL/6, OT-I RAG1⫺/⫺, and C57BL/6 Thy-1.1 mice were obtained from
Charles River Laboratories, Taconic Farms, and The Jackson Laboratory,
respectively. OT-I Thy1.1 mice were first generation crosses of OT-I
RAG1⫺/⫺ and C57BL/6 Thy-1.1 mice. All animals were maintained in
pathogen-free facilities. Recombinant vaccinia virus expressing OVA (vaccinia-OVA) was a gift from Dr. J. Yewdell (National Institute of Allergy
and Infectious Diseases, Bethesda, MD). Mice were immunized with 1 ⫻
106 PFU virus i.v. via the dorsal tail vein. All protocols were approved by
the Institutional Animal Care and Use Committee.
Adoptive transfer of OT-I cells
Mediastinal LN
Ax/Brach LN
Spleen
Mesenteric LN
0.6
43.1
0.2
44.3
0
13.2
43
3.3
52.2
3.7
90.5
3.8
91.8
0.2
55.5
0.3
6.4
0.1
3.2
0.1
11.3
2.0
42.4
3.3
90
2.2
94.5
2.5
86.1
0
1.5
0.1
2.8
0.1
1.7
95.2
2.3
94.8
2.5
95.7
5.8
0
4.4
IP
CD69
IV
0
0.6
AT
only
5.8
93.5
3.3
CFSE
FIGURE 1. BMDC immunization via different routes results in T cell
priming in distinct and limited lymphoid compartments. Mice received 4 ⫻
106 CD8-enriched, CFSE-labeled OT-I cells and were immunized 18 –36 h
later with CD40L-activated OVA257-pulsed BMDC or no BMDC (adoptive
transfer (AT) only) via the indicated route. Lymphoid organs were harvested 22 h later. Dot plots are gated on CD8⫹ H-2Kb-OVA257-tetramer⫹
lymphocytes. Data is representative of three independent experiments.
Flow cytometric analysis of surface markers and sorting
Single cell suspensions were incubated with anti-CD16/32 (93, eBioscience) to block Fc receptors. PerCP anti-CD8␣ (53-6.7), PE anti-␣4 integrin (R1–2), and PE anti-␣4␤7 (DATK32) were from BD Biosciences.
allophycocyanin anti-IFN-␥ (XMG1.2), allophycocyanin anti-Thy1.2 (53–
2.1) and anti-Thy1.1 (HIS51), PE anti-CD69 (H1.2F3), FITC and PE antiCD44 (IM7), PE and PE-Cy7 anti-CD62L (MEL-14), and anti-CD127-PE
(A7R34) were from eBioscience. Allophycocyanin-conjugated H-2KbOVA257 tetramer was generated in-house. Cells from mice treated with
anti-CD62L in vivo were blocked with 10% normal goat serum before
incubation with PE goat F(ab)⬘2 anti-rat IgG (Jackson ImmunoResearch),
followed by purified rat IgG (Jackson ImmunoResearch) and then PerCPCy5.5 anti-CD8␣ and allophycocyanin anti-Thy1.2. Samples were analyzed on FACSCalibur and FACSCanto instruments (Becton Dickinson)
using Flow Jo software (TreeStar). Electronic sorting was conducted on a
Becton Dickinson FACSVantage SE Turbo Sorter with DIVA Option.
Single cell suspensions from the spleen and pooled LN of OT-I RAG1⫺/⫺
and OT-I Thy1.1 mice were enriched for CD8 T cells by negative selection
(Stem Cell Technologies). Preparations were consistently 97–99% CD8⫹
by flow cytometry. CD8-enriched OT-I Thy1.1 cells were ⬎90% H-2KbOVA257 tetramer positive, at least 85% CD44low and CD62Lhigh, ⬎ 98%
CD69low, and ⬍ 2% ␣4high. In some cases, cells were labeled with 4 ␮M
CFSE (Molecular Probes) in PBS for 15 min at 37°C before injection.
Unless otherwise stated, 4 ⫻ 106 CD8-enriched OT-I cells were injected
i.v. into the dorsal tail vein of sex-matched recipients. In experiments involving transfer of redistributed OT-I cells, 2 ⫻ 104 electronically sorted
OT-I cells were injected into Thy1-mismatched naive recipients together
with 5 ⫻ 106 CFSE-labeled Thy1 host-matched T cells.
Lymphoid cells from mice 5 days after i.v. immunization were assessed for
cytokine production by incubation for 5 h at a ratio of 1:1 with LB15.13
stimulator cells that had been pulsed with 100 ␮M OVA257. Medium was
supplemented with 50 U/ml IL-2 (Chiron) and 10 ␮g/ml brefeldin A (Sigma-Aldrich). Lymphoid cells were pretreated with 100 ␮M TAPI-2 (Peptides International) for 1 h and during the assay to prevent CD62L downregulation as described (46). Cells were fixed and permeabilized using
Cytofix/Cytoperm (BD Biosciences) and stained for intracellular IFN-␥.
DC culture and immunizations
BMDC immunization leads to localized activation of CD8 T
cells followed by redistribution into noncontiguous LN
CD40L-activated BMDC were generated as previously described (44). Before injection, BMDC were pulsed with 10 ␮M OVA257 (corresponding to
residues 257–264 of chicken OVA) for 1 h at 37°C in the presence of 10
␮g/ml human ␤2-microglobulin (Calbiochem). Mice were immunized with
1 ⫻ 105 BMDC in 200 ␮l into the dorsal tail vein, the peritoneal cavity, or
the scapular fold.
FTY720 treatment and in vivo Ab blockade
Mice were injected i.p. with 1 mg/kg FTY720 (a gift of Dr. V. Brinkmann,
Novartis Pharma AG, Basel, Switzerland) in 200 ␮l 24 or 72 h after BMDC
injection and then every 24 h until harvest. Purified anti-CD62L (MEL-14,
American Type Culture Collection) (45) or rat IgG (Sigma-Aldrich) were
administered i.v. at 100 ␮g per mouse 6 h after BMDC injection and then
every 24 h until harvest.
Analysis of intracellular IFN-␥ production
Results
We previously demonstrated that peptide-pulsed BMDC infiltrate
a limited subset of lymphoid organs based on injection route, resulting in localized T cell activation in specific lymphoid compartments (39). CD8⫹ OT-I TCR transgenic T cells labeled with CFSE
were adoptively transferred into mice, and OVA257 peptide-pulsed
CD40L-activated BMDC were injected by either i.p., i.v., or s.c
routes 18 –36 h later. Twenty-two hours after BMDC injection,
activated OT-I cells were identified based on expression of CD69.
CD69⫹ OT-I cells were localized to spleen and mediastinal LN
after i.v. immunization, to mesenteric LN and mediastinal LN after
i.p. immunization, and to axillary/brachial LN after s.c immunization (Fig. 1 and data not shown). S.c. immunization also led to
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Unlike pathogens, exogenous bone marrow-derived DC
(BMDC) localization is highly restricted in vivo, and depends on
the injection route (32–39). Thus, BMDC enable immune responses in individual lymphoid compartments to be studied without the uncertainty of Ag localization and persistence. To date,
several studies have examined the characteristics of CD8 immune
responses that develop after BMDC immunization and have noted
differences based on route of injection (32, 37– 42). However, few
have examined the early temporal phases and spatial distributions
of these responses, and how these relate to the characteristics of the
resulting memory cells.
In the present work, we have used the localized trafficking of
BMDC to examine the activation and differentiation of CD8 T
cells in individual lymphoid compartments over time. Furthermore, we have confined these activated cells to lymphoid tissue by
using the drug FTY720 (43). Using these techniques, we have
identified a population of extensively divided cells that rapidly
redistribute into demonstrably Ag-free LN in a CD62L-dependent
manner. These redistributed cells exhibit effector characteristics,
and also contain memory precursors. The existence of this population is not currently appreciated in models of primary immune
responses or memory establishment. We suggest that redistribution
of activated cells to Ag-free LN early during the primary response
represents an important mechanism for establishment of LN resident TCM cells, and an important characteristic of central memory
precursor cells that is not included in current models.
1815
1816
CD8 T CELL REDISTRIBUTION TO LYMPH NODES
A
Mediastinal LN
*
Spleen
*
Ax/Brach LN
Inguinal LN
Mesenteric LN
96.3
97.2
87.8
87.4
91.8
98.0
99.1
30.0
32.3
45.4
Inguinal LN
Mesenteric LN
Cell Number
Saline
FTY720
CFSE
B
Mediastinal LN
Spleen
Ax/Brach LN
92.7
46.0
45.0
*84.6
99.0
34.3
8.7
18.7
95.2
Cell Number
Saline
FTY720
CFSE
C
Mediastinal LN
Spleen
Ax/Brach LN
72.9
89.9
*
96.0
*
37.2
76.8
8.9
18.3
95.3
8.51
3.7
Inguinal LN
Mesenteric LN
Cell Number
Saline
FTY720
CFSE
FIGURE 2. BMDC immunization leads to localized activation of CD8 T cells followed by redistribution into noncontiguous LN. Mice received
CD8-enriched, CFSE-labeled, Thy-mismatched OT-I cells and were immunized i.v. (A), i.p. (B), or s.c. (C) 18 –36 h later with CD40L-activated OVA257pulsed BMDC. FTY720 or saline treatment was initiated 24 h later and repeated every 24 h thereafter until harvest of lymphoid tissue at 72 h (A and B)
or 96 h (C) for flow cytometry. Histograms are gated on CD8⫹ H-2Kb-OVA257-tetramer⫹ (A and B), or CD8⫹ Thy1.1⫹ (C) lymphocytes and are
representative of five to six mice in three independent experiments. Numbers in plots indicate percentage of OT-I cells that have divided at least once.
Asterisks indicate priming compartments as defined by CD69 up-regulation in Fig. 1 and in the text.
activation of cells in spleen, although this was highly variable in
different experiments (data not shown). By 72–96 h after immunization, CFSE dilution revealed a substantial number of divided
OT-I cells in these sites of CD69 up-regulation, or “priming compartments” (indicated by asterisks in Fig. 2). However, extensively
divided OT-I cells were also present in all other LN examined at
this time. Greater than 90% of OT-1 cells remained undivided in
the same compartments of mice that had received either no BMDC
or unpulsed BMDC (data not shown), demonstrating that divided
cells in these other LN had been activated by Ag.
The presence of divided OT-I cells in LN other than those in
which initial BMDC priming occurred was not due to delayed
migration of BMDC to these sites: we previously demonstrated
that OT-I cells injected up to 48 h after BMDC still underwent
activation only in the previously identified priming compartments (39). Thus, the presence of divided OT-I cells in LN
noncontiguous with the priming compartments appeared to represent the dissemination of activated T cells. To directly test
this hypothesis, we confined OT-I cells to the compartment in
which they were activated by treating mice with FTY720, a
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
*88.8
The Journal of Immunology
Mediastinal LN
Spleen
*
*
72.4%
4x10
41.1%
63.2%
4
IV
CD62L
4x10
22.5%
12.4%
Spleen
*
Naive
25%
Mediastinal LN
76.9%
5
48.2%
4x10
B
Ax/Brach LN
ova257+H-2Kb tetramer
A
1817
*
0.008
0.0003
0.008
0.25
1.4
0.12
56.1%
CD8a
3
Mediastinal LN
Spleen
*
38%
*
8.7%
6.9%
19.2%
CD62L
4x10
7.6%
Ax/Brach LN
2
CD44
CFSE
D
Mediastinal LN
*
65.6
25.8
2.2
65.8
3.0
24.3
1.0
40.2
0.8
73.4
6.0
21.1
4.0
64.7
0.6
3.9
1.5
56%
Inguinal LN
8.3
63.8
56.8
34%
*
2.2
1.4
33.4
Ax/Brach LN
Spleen
71%
Mesenteric LN
11.3
60.2
20.7
2.4
29.9
82.2
9.2
58.9
2.5
73.2
13.7
2.7
29.2
1.6
22.7
76%
67%
7.9
CD62L
IV
2.0
AT
only
CFSE
FIGURE 3. Redistribution of divided T cells occurs at physiological T cell precursor frequencies. A and D, Thy1.1⫹ mice received the indicated number
(A) or 4 ⫻ 106 (D) CD8-enriched, CFSE-labeled Thy1.2⫹ OT-I cells and were immunized i.v. 24 h later with BMDC. Lymphoid organs were harvested
7 (A) or 3 days (D) after immunization. Dot plots are gated on CD8⫹ Thy1.2⫹ lymphocytes. Values in large type indicate the percentage of OT-I cells that
had divided one or more times and were CD62Lhigh. B and C, Endogenous responses were evaluated 7 days after immunization of normal C57BL/6 mice
with CD40L-activated OVA257-pulsed BMDC. Dot plots are gated on live (B) or CD8⫹ H-2Kb-OVA257-tetramer⫹ (C) lymphocytes. Values in bold indicate
the percentage of CD8⫹ cells that were H-2Kb-OVA257-tetramer⫹ (B) or the percentage of OT-I cells that had divided one or more times and were
CD62Lhigh (C). Data is representative of two to four mice per condition in at least three independent experiments. Asterisks indicate priming compartments
as defined by CD69 up-regulation in Fig. 1 and in the text.
pharmacological analog of sphingosine 1-phosphate that inhibits T lymphocyte egress from LN by binding to S1P1 receptors
(43). In the priming compartments of immunized mice treated
with FTY720, the CFSE dilution profiles showed an accentuated accumulation of more extensively divided cells compared
with controls (Fig. 2), and a 3– 4-fold increase in the average
number of activated OT-I cells in priming LN (data not shown).
However, FTY720 treatment reduced the presence of divided
OT-I cells in noncontiguous LN of these same mice by 60 –95%
in different experiments. These data demonstrate that divided
OT-I cells exited priming compartments and redistributed to
Ag-free LN by 3 days after immunization.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
C
18.1%
Ax/Brach LN
1818
CD8 T CELL REDISTRIBUTION TO LYMPH NODES
Redistribution of divided T cells occurs at physiological T cell
precursor frequencies
It was possible that the redistribution of activated T cells from
priming to Ag-free lymphoid compartments is due to the adoptive
transfer of large numbers (4 ⫻ 106) of T cells, resulting in the
“spillover” of activated T cells into other LN. Thus, we compared
responses elicited by i.v. immunization with BMDC in mice transferred with as few as 4 ⫻ 102 OT-I T cells. We also examined the
endogenous CD8 T cell response in mice that did not receive any
transferred cells. By day 7 after immunization, redistribution of
CFSE-diluted cells into axillary/brachial LN had occurred at all
adoptive transfer numbers tested (Fig. 3A). Similar data were also
obtained after immunization via the i.p. and s.c routes (data not
shown). Redistribution of activated OVA257⫹H-2Kb tetramer positive CD8 cells was also evident in the endogenous response (Fig.
3, B and C). These results demonstrate that the rapid redistribution
of activated T cells into Ag-free LN is not simply due to “spillover” from priming compartments based on limitations of space
and large numbers of activated cells, but is also characteristic of
immune responses based on relatively low numbers of T cells.
Activated CD62Lhigh OT-I cells redistribute into noncontiguous
LN predominantly via a CD62L-dependent mechanism
After immunization by different routes, a significant fraction of
divided OT-I cells in priming compartments of mice that received
4 ⫻ 106 OT-I cells had down-regulated CD62L expression by
72 h, consistent with a pre-effector or effector phenotype (Fig. 3D
and data not shown). However, ⬃1/3 of these divided cells remained CD62Lhigh despite having undergone as many as 6 –7 cell
divisions. Although the divided cells in Ag-free LN had undergone
a comparable number of cell divisions, the CD62Lhigh fraction was
even greater: 62–76% (Fig. 3D and data not shown). Similar data
were also obtained after immunization via the i.p. and s.c routes
(data not shown). In keeping with previous work (24, 47), we
found that the percentage of cells that remained CD62Lhigh after
activation was directly correlated with the number of adoptively
transferred cells (Fig. 3, A and C and data not shown). However,
in all cases, as well as in the endogenous response, the representation of CD62Lhigh divided cells was enhanced in Ag-free axillary
LN (Fig. 3, A and C). This again demonstrates that redistribution
is not due to the elevated percentage of activated CD62Lhigh cells
that develop in animals transferred with large numbers of OT-I
cells.
The above results suggested that CD62L was responsible for the
entry of divided OT-I cells into Ag-free LN. To test this, i.v. immunized animals were treated with anti-CD62L Ab beginning 6 h
after BMDC injection, and continuing every 24 h until harvest 3
days postimmunization. This resulted in the accumulation of more
extensively divided OT-I cells in the mediastinal LN priming compartment (Fig. 4A), and the ratio of divided to undivided cells
increased from ⬃5:1 to roughly 150:1. This likely reflects antiCD62L blockade of entry of naive cells coupled with continued
division of activated OT-I cells already in the LN. In contrast,
CD62L blockade substantially inhibited the redistribution of
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 4. Activated CD62Lhigh OT-I cells redistribute into noncontiguous LN predominantly via a CD62L-dependent mechanism. Mice received 4 ⫻
106 CD8-enriched, CFSE-labeled OT-I cells and were immunized i.v. with BMDC 18 –36 h later. A, B, and D, Anti-CD62L or Rat IgG was injected 6 h
later and every 24 h thereafter until harvest at 72 h. Dot plots and histogram are gated on CD8⫹ Thy1.2⫹ lymphocytes and are representative of four to
six mice in two to three independent experiments. CD62L expression on cells from anti-CD62L treated was measured as described in Materials and
Methods. B, Numbers in large type indicate percentage of divided cells expressing ␣4␤7. A and B are from independent experiments. Asterisks indicate
priming compartments as defined by CD69 up-regulation in Fig. 1 and in the text. C, CD62L expression on divided CD62Lhigh (black fill) and CD62Llow
(solid line) cells in axillary/brachial LN. Dashed line is negative staining control lacking only anti-CD62L Ab. D, Summary data showing mean values
(⫾SEM) from three mice in one experiment representative of two independent experiments.
The Journal of Immunology
1819
A
Mediastinal LN
78%
*
Spleen
Ax/Brach LN
*
Cervical LN
34.4%
79.5%
80%
30.8%
77.9%
80.8%
Ax/Brach LN
Cervical LN
CD62L
Saline
84.3%
FTY720
Day 3-9
CFSE
B
Mediastinal LN
Spleen
*
*
47%
20%
43%
43%
38%
27%
43%
65%
CD62L
Saline
FTY720
Day 3-9
CFSE
100
75
50
% CD62L
hi
(of activated OT-1)
C
Mediastinal LN
Spleen
25
Ax/Brach LN
*
*
Mesenteric LN
0
0
5
10
15
20
25
30
35
40
Day after immunization
divided OT-I cells into Ag-free peripheral LN (Fig. 4A). Although
there was seemingly little effect of anti-CD62L treatment on the
redistribution of divided OT-I cells into mesenteric LN, the majority of these cells in anti-CD62L-treated mice expressed ␣4␤7
integrin, while those in control mice were predominantly negative
(Fig. 4B). No such enrichment for ␣4␤7⫹ OT-I cells was evident
in other Ag-free LN. Expression of this integrin is known to suffice
for entry of naive or memory T cells into mesenteric LN (48).
Thus, our results show that activated cells that redistribute into
most Ag-free LN do so by a CD62L-dependent mechanism.
The CD62L-dependent redistribution of CD62Lhigh divided
OT-I cells was not surprising. Importantly, however, the level of
CD62L expression on CD62Llow cells was significantly above
background (Fig. 4C). Thus, we were interested to know whether
CD62Llow cells, which predominated at lower adoptive transfer
numbers and in the endogenous response, redistributed by this
same mechanism. To test whether CD62Llow cells were enriched
among the divided OT-I cells that continued to redistribute in antiCD62L treated mice, we used fluorescently labeled anti-rat IgG to
detect bound anti-CD62L Ab. Using this approach, we could
clearly distinguish CD62Lhigh and CD62Llow populations in all
lymphoid organs. We found that there was no enrichment of
CD62Llow divided OT-I cells in Ag-free axillary/brachial or cervical LN in anti-CD62L treated mice relative to the populations in
untreated mice (Fig. 4, A and D). Thus, the presence of divided
CD62Lhigh and CD62Llow cells in Ag-free peripheral LN is
equally dependent on CD62L. This also establishes that, in
contrast to redistribution into mesenteric LN, CD62Llow cells have
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 5. Stability of CD62Lhigh and CD62Llow
cells in Ag-free LN after redistribution. Thy1.1⫹ mice
received 4 ⫻ 106 (A and C) or 4 ⫻ 104 (B) CD8-enriched, CFSE-labeled Thy1.2⫹ OT-I cells and were immunized i.v. with BMDC 24 h later. A and B, FTY720 or
vehicle treatment was initiated 72 h later and repeated
every 24 h thereafter until harvest at 9 days. A and B,
Dotplots are gated on CD8⫹ Thy1.2⫹ lymphocytes. C,
Proportion of Ag-experienced (CD44high or having undergone ⱖ1 cell division) OT-1 cells in lymphoid compartments that maintained CD62Lhigh expression after
BMDC immunization. Asterisks indicate priming compartments as defined by CD69 up-regulation in Fig. 1
and in the text.
1820
CD8 T CELL REDISTRIBUTION TO LYMPH NODES
Redistributed OT-I cells have a well-differentiated effector cell
phenotype
Most models of CD8 T cell differentiation envision that cells with
effector activity will migrate from lymphoid sites to peripheral
nonlymphoid tissues, blood, and spleen. Thus, it was of interest to
determine the effector status of the divided CD62Lhigh and
CD62Llow OT-I cells that had redistributed into Ag-free LN.
Therefore, we assessed their ability to produce IFN-␥ after a short
ex vivo peptide restimulation. To avoid the short-term reduction in
CD62L expression that follows Ag stimulation, we incubated cells
with TAPI-2, an inhibitor of the TNF-␣-converting enzyme sheddase (46), during stimulation. TAPI-2 treatment had no effect on
IFN-␥ production (data not shown). Compared with divided cells
in spleen as representative of fully differentiated effectors, we
found no significant difference in the percentage of divided cells in
Ag-free LN making IFN-␥, or in the mean fluorescence intensity of
IFN-␥ expression (Fig. 6, A–F). Interestingly, in both Ag-free LN
and spleen, the representation of effector cells in the CD62Lhigh
subset was equivalent to or higher than that of the CD62Llow subset, depending on the number of cells that were adoptively transferred. Divided cells in Ag-free LN and spleen also uniformly
down-regulated CD127 ⫻ 72 h postimmunization (Fig. 6G), and
up-regulated CD44 and ␣4 integrin to similar extents by 6 days
after i.v. immunization (data not shown). Similar data were also
obtained after immunization via the i.p. and s.c routes (data not
shown). Thus, divided OT-I cells that had redistributed to peripheral LN were well differentiated.
Redistributed OT-I cells contain memory cell precursors
The foregoing results established that the divided OT-I cells that
had redistributed to peripheral LN persisted for at least 9 days, but
also exhibited characteristics of well-differentiated effector cells.
Thus, we were interested in whether these cells were terminal, or
were also a source of memory. In keeping with the latter possibil-
4
4x10 OT-I
Divided OT-I
Divided CD62Lhi
Divided CD62Llo
50
40
%IFN-γ Positive
40
30
20
10
30
20
10
0
0
Ag-free LN
Ag-free LN
Spleen
Spleen
*
*
C
D
6
4x10 OT-I
Ag-free LN
4
4x10 OT-I
Ag-free LN
Spleen
Spleen
*
35.3
33.3
37.3
28.1
CD62L
*
28.5
21.2
17.2
13.7
IFN-γ
E
IFN-γ
F
6
4x10 OT-I
105
Divided CD62Lhi
Divided CD62Llo
104
103
10
2
Ag-free LN Spleen
4
4x10 OT-I
105
Divided CD62Lhi
Divided CD62Llo
104
103
10
2
Ag-free LN Spleen
*
G
*
Mediastinal LN
Spleen
*
*
14.6
2.5
7.5
15.3%
81
Ax/Brach LN
4.6
7.3
84.7
8.2
3.2
63.8
15.4
10.5%
10.3%
8.1%
1.9
19.2
Mesenteric LN
9.7
69.7
6.7
CFSE
FIGURE 6. Redistributed OT-I cells have characteristics of well-differentiated effectors. Thy1.1⫹ mice received Thy1.2⫹ OT-I cells and were
immunized i.v. 24 h later with BMDC, and the indicated lymphoid tissues
were harvested 3 (G), 5 (A, C, and E), or 7 (B, D, and F) days later. A–F,
Splenocytes or LN cells were assayed for Ag-induced production of intracellular IFN-␥ as described in Materials and Methods. Dot plots are gated
on divided CD8⫹ Thy1.2⫹ lymphocytes. Large type numbers in right quadrants indicate percentages of either CD62Lhigh or CD62Llow cells producing IFN-␥. Data are representative of eight mice in three independent experiments (A), or seven mice in two independent experiments (B). Bar
graphs represent summary data from one experiment with three immunized
mice and two unimmunized mice (A and C) or four immunized mice (B and
D) indicating mean (⫾SEM) percentage (A and B) or geometric mean
(⫾SEM) fluorescence intensity (E and F) of IFN-␥⫹ cells. G, CD127 staining 3 days after immunization. Dot plots are gated on CD8⫹Thy1.2⫹ lymphocytes. Large type numbers in upper left indicate percentage of divided
cells expressing CD127. Data are representative of eight mice in three
independent experiments. Asterisks indicate priming compartments as defined by CD69 up-regulation in Fig. 1 and in the text.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
To determine whether CD62Lhigh cells down-regulated this molecule after redistribution into peripheral LN, we treated i.v. immunized mice with FTY720 starting on day 3 and every 24 h
thereafter until harvest on day 9. By delaying FTY720 treatment,
we allowed initial redistribution to occur, but then trapped redistributed cells and prevented movement of cells between compartments. Using mice transferred with either 4 ⫻ 106 or 4 ⫻ 104 OT-I
cells, we found that the ratios of divided CD62Lhigh to CD62Llow
OT-I cells in Ag-free LN of FTY720 treated mice were not different from the ratios in untreated mice (Fig. 5, A and B). This
result demonstrates that neither population expands or contracts in
relation to the other during the 6 days after redistribution occurs.
Consistent with this, in the absence of FTY720 treatment the relative percentages of CD62Lhigh and CD62Llow cells in Ag-free LN
of i.v immunized animals do not change between 7 and 40 days
after immunization (Fig. 5C). Similar data were also obtained after
immunization via the i.p. route (data not shown). We cannot rigorously exclude the possibility that conversion of CD62Lhigh cells
occurs, but is balanced by an equivalent conversion of CD62Llow
cells to a CD62Lhigh phenotype. Nonetheless, these data collectively indicate that both CD62Lhigh and CD62Llow CD8 T cells are
long-term residents of LN.
%IFN-γ Positive
cells in Ag-free LN after
CD62L
and CD62L
Unimmunized OT-I
Divided OT-I
Divided CD62Lhi
Divided CD62Llo
50
Mean Fluorescence Intensity of IFN-γ
Stability of CD62L
redistribution
low
B
6
4x10 OT-I
CD127
high
A
Mean Fluorescence Intensity of IFN-γ
no method to enter peripheral LN independent of CD62L. This
result suggests that either the level of expression on CD62Llow
cells suffices for LN entry or these cells enter LN as CD62Lhigh
cells and down-regulate it thereafter.
The Journal of Immunology
1821
ity, we found a significant increase in the percentage of divided
OT-I cells in Ag-free LN that were CD127⫹ by 7 days after immunization (Fig. 7A). Even larger percentages of these cells were
CD127⫹ on day 7 when the adoptive transfer number was reduced
(Fig. 7B). Although these results were consistent with the idea that
the redistributed T cells re-expressed CD127, they did not exclude
the possibility that CD127⫹ cells entered Ag-free LN at a later
time point. However, when we trapped redistributed cells in Agfree LN by initiating FTY720 treatment on day 3 and continuing
until day 7, we found that over two-thirds of the divided T cells in
Ag-free axillary/brachial and cervical LN were CD127⫹ (Fig. 7C).
This result indicates that the divided OT-I cells that redistribute on
day 3 posses the ability to up-regulate CD127 by day 7, or that a
minor subset of redistributed cells that are initially CD127⫹ expand significantly over this time period. Either mechanism is con-
sistent with the idea that the redistributed cells contain memory
CD8 T cell precursors.
To directly establish the potential of redistributed cells to seed
memory, we sorted divided CD62Lhigh and CD62Llow Thy1.1⫹
OT-I T cells from Ag-free LN 3 days after i.v. immunization. The
purity of each of these populations was ⬎90% as assessed by flow
cytometry (data not shown). Twenty thousand cells of each phenotype were transferred into naive Thy1-mismatched hosts together with a known quantity of CFSE-labeled naive T cells obtained from a TCR transgenic mouse with an irrelevant specificity
(49) and Thy1-matched to the recipient. These cells served as an
internal control for the efficiency of transfer in different recipients.
Three weeks after transfer, we immunized some of the recipients
with recombinant vaccinia expressing OVA and analyzed the representation of OT-I cells in LN and spleen 7 days later. Transferred
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 7. Redistributed OT-I cells contain memory cell precursors. Thy 1.1⫹ mice were adoptively transferred with 4 ⫻ 106 (A and C), or the indicated
number of CFSE-labeled Thy 1.2⫹ OT-I cells and immunized i.v. with BMDC 18 –36 h later. Lymphoid organs were harvested at the indicated times after
immunization (A), or at 7 days (B and C). A, Mean values from two to six mice (⫾SEM) pooled from seven independent experiments. B, Mean values
(⫾SEM) from three mice per group in one experiment. C, Mice received FTY720 or vehicle beginning 3 days after immunization and then every 24 h until
harvest on day 7. Dot plots are gated on CD8⫹ Thy1.2⫹ lymphocytes. Large type numbers in upper left indicate percentage of divided cells expressing
CD127. D, Pooled Ag-free LN from Thy 1.2⫹ mice that had received Thy1.1⫹ OT-I cells were harvested 72 h after i.v. immunization. Cells were
electronically sorted to select for CFSE dilution (more than one division), 7-AADneg, CD8⫹, and Thy1.1⫹. CD62Lhigh and CD62Llow subsets were collected
separately. Sorted cells (2 ⫻ 104) were transferred into naive Thy1.2⫹ mice together with 3 ⫻ 106 CFSE-labeled Thy1.2⫹ TCR transgenic T cells with
an irrelevant specificity (49). These were used as an internal control for differences in injection efficiency and cellular distribution among mice. Three weeks
later, mice were challenged with vaccinia-OVA i.v. or left unchallenged and lymphoid organs were harvested on day 7 after infection. Three to four million
events were collected in each sample. Numbers indicate gated populations as a percentage of total lymphocytes. Asterisks indicate priming compartments
as defined by CD69 up-regulation in Fig. 1 and in the text.
1822
OT-I cells were virtually undetectable in either peripheral LN or
spleen of unimmunized animals (Fig. 6D). However, they were
readily detectable in both compartments of vaccinia-challenged
animals. Relative to the cotransferred CFSE-labeled control cells,
recall OT-I cells were more evident in spleen that LN, as would be
expected for day 7 effectors. Nonetheless, both CD62Lhigh and
CD62Llow transferred cells gave rise to significant numbers of recall cells in LN. Interestingly, the number of recall cells derived
from transferred CD62Lhigh precursors was ⬃5-fold higher than
from the same number of CD62Llow precursors. Collectively, our
results suggest that the activated cells that redistribute into Ag-free
LN within 72 h after activation contain precursors of memory CD8
cells, and these are enriched in the subset of redistributed cells that
are CD62Lhigh.
Discussion
tivated OT-I cells redistribute from priming compartments to Agfree LN. We have extended the observations of Liu et al. (50) by
showing that redistribution occurs regardless of the number of
OT-I cells adoptively transferred, and also during the endogenous
response, indicating that it is not simply a consequence of activating large numbers of Ag-specific T cells in spatially limited lymphoid compartments. We have also observed FTY720-inhibitable
redistribution of activated T cells to LN using vaccinia virus as an
immunogen (our unpublished data) demonstrating that it is not
limited to responses initiated with BMDC. Instead, redistribution
of activated CD8 T cells to Ag-free LN is a normal aspect of
immune responses whose significance has not been well
understood.
Our work has also demonstrated that redistribution of activated
CD8 T cells to most peripheral Ag-free LN is mediated by CD62L,
in keeping with its already well-known involvement in the entry of
naive T cells into LN. In separate work (39), we established that a
small population of activated CD8 T cells expressing ␣4␤7 redistribute from mesenteric to mediastinal LN in a process mediated
by that integrin. However, in that instance, both LN were priming
compartments. In the present study, we observed that activated
␣4␤7⫹ cells redistribute to Ag-free mesenteric LN in a CD62Lindependent manner. Thus, redistribution of activated cells to Agfree LN during the primary immune response is a general phenomenon that can be mediated by distinct mechanisms.
Interestingly, regardless of the number of cells initially transferred,
redistributed CD8 cells included subsets expressing high and low
levels of CD62L, and the fraction of CD62Lhigh cells was higher
in cells that had redistributed than in priming LN. However, the
representation of CD62Lhigh and CD62Llow cells in Ag-free LN
was equally susceptible to anti-CD62L blockade. The relative frequencies of these populations also remained stable after redistribution, suggesting that they do not interconvert. However, it remains possible that a portion of redistributing cells down-regulate
CD62L very rapidly after LN entry, and also that this process is
subsequently balanced by up-regulation, leading to the appearance
that CD62Lhigh and CD62Llow cells are stable and separate populations. Alternatively, CD62Llow expression, which is measurably above that of CD62Lneg cells, may be sufficient for LN entry.
Regardless of the exact mechanism, we and others (24, 50) have
also observed that previously activated CD8 cells with a reduced
level of CD62L expression persist in LN long term.
Recent work has led to significant concerns about how accurately the processes of differentiation and memory are represented
in animals transferred with large numbers of CD8 T cells. In particular, it was demonstrated that the fraction of activated CD8 cells
that retain CD62Lhigh expression after infection with an Ag expressing pathogen is higher in animals transferred with relatively
high numbers of cells (24, 47). It was also shown that long-term
CD62Llow memory cells rescued from such animals reacquired a
CD62Lhigh phenotype after adoptive transfer, leading to their designation as “transitional memory cells” (24). The development of
transitional memory cells was diminished by increasing the number of endogenous APC, suggesting that an elevated ratio of Agspecific T cells to APC results in compromised or altered activation. Whether activated early stage CD62Llow cells show a similar
reacquisition of CD62L is unknown. However, asymmetrical division of a single T cell has been shown to produce memory and
effector lineage cells that are CD62Lhigh and CD62Llow, respectively, as early as the first cell division (51). Thus, while adoptive
transfer may alter the fraction of CD62Lhigh and CD62Llow expressing cells, both are physiologically relevant offspring to the
CD8 T cell activation process. Larger adoptive transfer numbers
were also associated with a higher fraction of activated early stage
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
In the current work, we have used local immunization with peptide-pulsed, CD40L-activated BMDC to characterize the differentiation of CD8 T cells in individual LN and their migration among
LN. Peptide-pulsed BMDC traffic rapidly into lymphoid tissue in
a highly restricted pattern after injection (37, 38), and remain localized and active for at least 48 h (39). Thus, the use of this
immunogen avoids the uncertainty about location and amount of
Ag that is inherent in the use of viruses, bacteria, or peptides in
adjuvant to study T cell activation in vivo. Using this system, we
have demonstrated the existence of a subset of activated CD8 T
cells that: 1) redistribute into Ag-free LN within 72 h via a
CD62L-dependent mechanism, 2) exhibit the characteristics of
fully differentiated effector cells; and 3) contain precursors of
memory cells.
An important observation in this study is that CD8 T cells activated in one LN redistribute within 3 days into other LN that are
demonstrably free of APC-bearing cognate Ag. Most models of
CD8 T cell differentiation emphasize its occurrence in lymphoid
compartments in which Ag is encountered, followed by dissemination to blood, spleen, and nonlymphoid tissues (5–14). However,
models for the differentiation of CD8 T cells in LN specifically
have evolved from studies in which differentiated effector cells
were identified primarily or exclusively in draining LN or spleen,
and presumptive nondraining LN were not evaluated (6, 15–18). It
was suggested in one previous study (20) that CD8 T cells activated after intranasal administration of influenza virus redistributed to nondraining LN. However, no conclusive data to support
this suggestion was provided, and it was also acknowledged that
these results might be due instead to delayed Ag presentation in
these initially nondraining compartments. Indeed, using the same
virus and route of administration, another group made similar observations and concluded that the response was “broadly systemic”
(19). This is in keeping with the demonstration that Ag presentation after viral infection is prolonged (21, 22) and occurs in LN
where virus is not detected (15). The suggestion that activated CD8
T cells had redistributed from priming to Ag-free LN was also
made in one prior study using BMDC as an immunogen (40).
Again, however, no direct demonstration of this phenomenon was
provided, and the authors acknowledged the possibility of disseminated Ag presentation due to transfer of peptide-MHC complexes
to endogenous DC. More recently, Liu et al. (50) used FTY720
administration to provide the first direct demonstration that virus
specific cells in skin-draining LN could redistribute to other LN.
However, this study did not investigate the mechanism of redistribution, nor the relationship of these cells to either conventional
effector cells or memory precursors. In this study, we circumvented uncertainty concerning Ag localization by using peptidepulsed BMDC, and used FTY720 to directly demonstrate that ac-
CD8 T CELL REDISTRIBUTION TO LYMPH NODES
The Journal of Immunology
proliferate in response to the vaccinia challenge. This possibility is
also consistent with other work (10, 11, 29, 31). Regardless of the
exact mechanism, our results suggest that early stage redistributed
CD62Lhigh cells are enriched for central memory precursors.
CD62Llow cells also harbor memory precursors, although these
may represent central, transitional, or effector memory lineages.
High and low level expression of CD62L is often used, irrespective of location, to define TCM and effector T memory subsets,
respectively (6, 31). However, the stable representation of both
CD62Lhigh and CD62Llow Ag experienced OT-I cells in LN for at
least 40 days, and the fact that both populations contain memory
precursors that give rise to recall responses in LN after adoptive
transfer, indicates that residence in an Ag-free LN is a more inclusive definition of TCM. In relation to this, we also suggest that
the rapid redistribution of CD8 T cells to Ag-free LN represents an
additional marker of TCM precursors, and is a process that leads to
the establishment of systemic memory.
Acknowledgments
We thank Sarah T. Lewis for generation of H-2Kb-OVA257 tetramer and
purification of anti-CD62L Ab. We also thank Janet V. Gorman for maintenance of the animal colony and Michael Solga for flow sorting.
Disclosures
The authors have no financial conflict of interest.
References
1. Bousso, P., and E. Robey. 2003. Dynamics of CD8⫹ T cell priming by dendritic
cells in intact lymph nodes. Nat. Immunol. 4: 579 –585.
2. Mempel, T. R., S. E. Henrickson, and U. H. von Andrian. 2004. T-cell priming
by dendritic cells in lymph nodes occurs in three distinct phases. Nature 427:
154 –159.
3. Miller, M. J., O. Safrina, I. Parker, and M. D. Cahalan. 2004. Imaging the single
cell dynamics of CD4⫹ T cell activation by dendritic cells in lymph nodes. J. Exp.
Med. 200: 847– 856.
4. Itano, A. A., S. J. McSorley, R. L. Reinhardt, B. D. Ehst, E. Ingulli,
A. Y. Rudensky, and M. K. Jenkins. 2003. Distinct dendritic cell populations
sequentially present antigen to CD4 T cells and stimulate different aspects of
cell-mediated immunity. Immunity 19: 47–57.
5. Flynn, K. J., G. T. Belz, J. D. Altman, R. Ahmed, D. L. Woodland, and
P. C. Doherty. 1998. Virus-specific CD8⫹ T cells in primary and secondary
influenza pneumonia. Immunity 8: 683– 691.
6. Masopust, D., V. Vezys, A. L. Marzo, and L. Lefrancois. 2001. Preferential
localization of effector memory cells in nonlymphoid tissue. Science 291:
2413–2417.
7. Wiley, J. A., R. J. Hogan, D. L. Woodland, and A. G. Harmsen. 2001. Antigenspecific CD8⫹ T cells persist in the upper respiratory tract following influenza
virus infection. J. Immunol. 167: 3293–3299.
8. Reinhardt, R. L., A. Khoruts, R. Merica, T. Zell, and M. K. Jenkins. 2001. Visualizing the generation of memory CD4 T cells in the whole body. Nature 410:
101–105.
9. Lefrancois, L., and D. Masopust. 2002. T cell immunity in lymphoid and nonlymphoid tissues. Curr. Opin. Immunol. 14: 503–508.
10. Zimmerman, C., K. Brduscha-Riem, C. Blaser, R. M. Zinkernagel, and
H. Pircher. 1996. Visualization, characterization, and turnover of CD8⫹ memory
T cells in virus-infected hosts. J. Exp. Med. 183: 1367–1375.
11. Busch, D. H., I. M. Pilip, S. Vijh, and E. G. Pamer. 1998. Coordinate regulation
of complex T cell populations responding to bacterial infection. Immunity 8:
353–362.
12. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac,
J. D. Miller, J. Slansky, and R. Ahmed. 1998. Counting antigen-specific CD8 T
cells: a reevaluation of bystander activation during viral infection. Immunity 8:
177–187.
13. Butz, E. A., and M. J. Bevan. 1998. Massive expansion of antigen-specific CD8⫹
T cells during an acute virus infection. Immunity 8: 167–175.
14. Badovinac, V. P., B. B. Porter, and J. T. Harty. 2002. Programmed contraction of
CD8⫹ T cells after infection. Nat. Immunol. 3: 619 – 626.
15. Usherwood, E. J., T. L. Hogg, and D. L. Woodland. 1999. Enumeration of antigen-presenting cells in mice infected with Sendai virus. J. Immunol. 162:
3350 –3355.
16. Hogan, R. J., W. Zhong, E. J. Usherwood, T. Cookenham, A. D. Roberts, and
D. L. Woodland. 2001. Protection from respiratory virus infections can be mediated by antigen-specific CD4⫹ T cells that persist in the lungs. J. Exp. Med.
193: 981–986.
17. Masopust, D., V. Vezys, E. J. Usherwood, L. S. Cauley, S. Olson, A. L. Marzo,
R. L. Ward, D. L. Woodland, and L. Lefrancois. 2004. Activated primary and
memory CD8 T cells migrate to nonlymphoid tissues regardless of site of activation or tissue of origin. J. Immunol. 172: 4875– 4882.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
cells that retained CD127 (47), although a second study using a
distinct immunogen observed different kinetics of CD127 loss and
re-expression, and a more complex relationship to adoptive transfer number (52).
We have demonstrated a direct correlation between adoptive
transfer number and CD62Lhigh expression on activated early stage
cells similar to that described previously (24, 47). However, we
found that the percentage of activated cells expressing CD127 on
day 7 was elevated, not diminished, when lower numbers of cells
were transferred. This difference with previous work is likely ascribable to our use of BMDC as an immunogen. The administration of exogenous DC may reduce the elevated T cell:APC ratio
thought to be responsible for altered T cell differentiation in other
models using pathogens presented by endogenous DC. Finally,
larger adoptive transfer numbers were associated with a smaller
fraction of cells expressing granzyme B, but a larger fraction expressing IL-2 and TNF (47). In our hands, we found that the fraction of cells expressing IFN-␥ was enhanced at high adoptive
transfer numbers. Nonetheless, in both studies, these differences
were quantitative rather than qualitative. Thus, although adoptive
transfer number can clearly affect the proportions of cells expressing effector or memory markers, it does not obviate the fundamental observation that activated CD8 T cells that have redistributed to
a Ag-free LN contain both effectors and memory precursors.
Another important observation in the present study is that the
CD8 cells that redistribute to Ag-free LN, regardless of their level
of CD62L expression, have characteristics of well-differentiated
effectors. Their production of IFN-␥ was equivalent to that of
“classical” splenic effectors, and they had acquired high level expression of either ␣4␤1 or ␣4␤7 integrin (39), enabling them to
home to peripheral nonlymphoid tissue. The biological significance of having CD8 T cells with effector activity in LN where
there is no Ag or active infection is not clear. In contrast, we have
also demonstrated that redistributed CD8 cells are not simply terminal effectors that die as the primary response contracts, but contain the precursors of memory cells. Regardless of adoptive transfer number, a significant fraction of redistributed cells express
CD127 by day 7, which has been associated with a memory or
prememory phenotype (25, 53). More directly, redistributed cells
rescued from LN 3 days after immunization persisted as memory
cells after adoptive transfer into naive recipients. This time frame
for the establishment of memory precursors is consistent with that
of other work in which activated cells were recovered from priming LN (18). From this perspective, the observation of effector
activity in the cells that redistribute to Ag-free LN is also consistent with other work suggesting that memory cells go through a
stage in which they express effector activity (18, 25, 29).
Interestingly, the size of the recall response was ⬃5-fold higher
in animals that had received redistributed OT-I cells with a
CD62Lhigh phenotype than in those receiving and equivalent number of CD62Llow cells. One possibility is that this reflects enhanced
survival or homeostatic proliferation of CD62Lhigh cells after
adoptive transfer (24). However, the ratio of these two populations
did not change from day 3 to 9 in the LN of animals treated with
FTY720, nor in the LN of untreated animals up to 40 days after
immunization. Alternatively, it is possible that the CD62Lhigh cells
distribute more efficiently into lymphoid tissue after adoptive
transfer, while the CD62Llow cells are more likely to enter peripheral tissue, and that this alters the extent to which they engage with
DC after vaccinia infection. This is in keeping with other work on
the distribution of adoptively transferred CD62Lhigh and
CD62Llow cells (6, 54, 55), although the cells used were usually
propagated in vitro or isolated from spleen rather than LN. Finally,
it is possible that that the CD62Lhigh cells have greater capacity to
1823
1824
37. Mullins, D. W., S. L. Sheasley, R. M. Ream, T. N. Bullock, Y. X. Fu, and
V. H. Engelhard. 2003. Route of immunization with peptide-pulsed dendritic
cells controls the distribution of memory and effector T cells in lymphoid tissues
and determines the pattern of regional tumor control. J. Exp. Med. 198:
1023–1034.
38. Mullins, D. W., and V. H. Engelhard. 2006. Limited infiltration of exogenous
dendritic cells and naive T cells restricts immune responses in peripheral lymph
nodes. J. Immunol. 176: 4535– 4542.
39. Sheasley-O’Neill, S. L., C. C. Brinkman, A. R. Ferguson, M. C. Dispenza, and
V. H. Engelhard. 2007. Dendritic cell immunization route determines integrin
expression and lymphoid and nonlymphoid tissue distribution of CD8 T cells.
J. Immunol. 178: 1512–1522.
40. Lambrecht, B. N., R. A. Pauwels, and G. B. St. de Fazekas. 2000. Induction of
rapid T cell activation, division, and recirculation by intratracheal injection of
dendritic cells in a TCR transgenic model. J. Immunol. 164: 2937–2946.
41. Dudda, J. C., J. C. Simon, and S. Martin. 2004. Dendritic cell immunization route
determines CD8⫹ T cell trafficking to inflamed skin: role for tissue microenvironment and dendritic cells in establishment of T cell-homing subsets. J. Immunol. 172: 857– 863.
42. Dudda, J. C., A. Lembo, E. Bachtanian, J. Huehn, C. Siewert, A. Hamann,
E. Kremmer, R. Forster, and S. F. Martin. 2005. Dendritic cells govern induction
and reprogramming of polarized tissue-selective homing receptor patterns of T
cells: important roles for soluble factors and tissue microenvironments. Eur.
J. Immunol. 35: 1056 –1065.
43. Brinkmann, V., and K. R. Lynch. 2002. FTY720: targeting G-protein-coupled
receptors for sphingosine 1-phosphate in transplantation and autoimmunity. Curr.
Opin. Immunol. 14: 569 –575.
44. Bullock, T. N. J., T. A. Colella, and V. H. Engelhard. 2000. The density of
peptides displayed by dendritic cells affects immune responses to human tyrosinase and gp100 in HLA-A2 transgenic mice. J. Immunol. 164: 2354 –2361.
45. Gallatin, W. M., I. L. Weissman, and E. C. Butcher. 1983. A cell-surface molecule involved in organ-specific homing of lymphocytes. Nature 304: 30 –34.
46. Jabbari, A., and J. T. Harty. 2006. Simultaneous assessment of antigen-stimulated
cytokine production and memory subset composition of memory CD8 T cells.
J. Immunol. Methods 313: 161–168.
47. Badovinac, V. P., J. S. Haring, and J. T. Harty. 2007. Initial T cell receptor
transgenic cell precursor frequency dictates critical aspects of the CD8⫹ T cell
response to infection. Immunity 26: 827– 841.
48. Wagner, N., J. Lohler, T. F. Tedder, K. Rajewsky, W. Muller, and D. A. Steeber.
1998. L-selectin and ␤7 integrin synergistically mediate lymphocyte migration to
mesenteric lymph nodes. Eur. J. Immunol. 28: 3832–3839.
49. Nichols, L. A., Y. Chen, T. A. Colella, C. L. Bennet, B. E. Clausen, and
V. H. Engelhard. 2007. Deletional self-tolerance to a melancyte/melanoma antigen derived from tyrosinase is mediated by a radio-resistant cell in peripheral and
mesenteric lymph nodes. J. Immunol. 179: 993–1003.
50. Liu, L., R. C. Fuhlbrigge, K. Karibian, T. Tian, and T. S. Kupper. 2006. Dynamic
programming of CD8⫹ T cell trafficking after live viral immunization. Immunity
25: 511–520.
51. Chang, J. T., V. R. Palanivel, I. Kinjyo, F. Schambach, A. M. Intlekofer,
A. Banerjee, S. A. Longworth, K. E. Vinup, P. Mrass, J. Oliaro, et al. 2007.
Asymmetric T lymphocyte division in the initiation of adaptive immune responses. Science 315: 1687–1691.
52. Joshi, N. S., W. Cui, A. Chandele, H. K. Lee, D. R. Urso, J. Hagman, L. Gapin,
and S. M. Kaech. 2007. Inflammation directs memory precursor and short-lived
effector CD8⫹ T cell fates via the graded expression of T-bet transcription factor.
Immunity 27: 281–295.
53. Schluns, K. S., W. C. Kieper, S. C. Jameson, and L. Lefrancois. 2000. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nat.
Immunol. 1: 426 – 432.
54. Weninger, W., M. A. Crowley, N. Manjunath, and U. H. von Andrian. 2001.
Migratory properties of naive, effector, and memory CD8⫹ T cells. J. Exp. Med.
194: 953–966.
55. Campbell, D. J., G. F. Debes, B. Johnston, E. Wilson, and E. C. Butcher. 2003.
Targeting T cell responses by selective chemokine receptor expression. Semin.
Immunol. 15: 277–286.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
18. Kedzierska, K., J. Stambas, M. R. Jenkins, R. Keating, S. J. Turner, and
P. C. Doherty. 2007. Location rather than CD62L phenotype is critical in the
early establishment of influenza-specific CD8⫹ T cell memory. Proc. Natl. Acad.
Sci. USA 104: 9782–9787.
19. Roman, E., E. Miller, A. Harmsen, J. Wiley, U. H. von Andrian, G. Huston, and
S. L. Swain. 2002. CD4 effector T cell subsets in the response to influenza:
heterogeneity, migration, and function. J. Exp. Med. 196: 957–968.
20. Lawrence, C. W., and T. J. Braciale. 2004. Activation, differentiation, and migration of naive virus-specific CD8⫹ T cells during pulmonary influenza virus
infection. J. Immunol. 173: 1209 –1218.
21. Jelley-Gibbs, D. M., D. M. Brown, J. P. Dibble, L. Haynes, S. M. Eaton, and
S. L. Swain. 2005. Unexpected prolonged presentation of influenza antigens promotes CD4 T cell memory generation. J. Exp. Med. 202: 697–706.
22. Zammit, D. J., D. L. Turner, K. D. Klonowski, L. Lefrancois, and L. S. Cauley.
2006. Residual antigen presentation after influenza virus infection affects CD8 T
cell activation and migration. Immunity 24: 439 – 449.
23. Kaech, S. M., S. Hemby, E. Kersh, and R. Ahmed. 2002. Molecular and functional profiling of memory CD8 T cell differentiation. Cell 111: 837– 851.
24. Marzo, A. L., K. D. Klonowski, B. A. Le, P. Borrow, D. F. Tough, and
L. Lefrancois. 2005. Initial T cell frequency dictates memory CD8⫹ T cell lineage commitment. Nat. Immunol. 6: 793–799.
25. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, and
R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies
effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4:
1191–1198.
26. Chang, J. T., V. R. Palanivel, I. Kinjyo, F. Schambach, A. M. Intlekofer,
A. Banerjee, S. A. Longworth, K. E. Vinup, P. Mrass, J. Oliaro, N. Killeen, et al.
2007. Asymmetric T lymphocyte division in the initiation of adaptive immune
responses. Science 315: 1687–1691.
27. Manjunath, N., P. Shankar, J. Wan, W. Weninger, M. A. Crowley, K. Hieshima,
T. A. Springer, X. Fan, H. Shen, J. Lieberman, and U. H. von Andrian. 2001.
Effector differentiation is not prerequisite for generation of memory cytotoxic T
lymphocytes. J. Clin. Invest. 108: 871– 878.
28. Baron, V., C. Bouneaud, A. Cumano, A. Lim, T. P. Arstila, P. Kourilsky,
L. Ferradini, and C. Pannetier. 2003. The repertoires of circulating human CD8⫹
central and effector memory T cell subsets are largely distinct. Immunity 18:
193–204.
29. Wherry, E. J., V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia,
U. H. von Andrian, and R. Ahmed. 2003. Lineage relationship and protective
immunity of memory CD8 T cell subsets. Nat. Immunol. 4: 225–234.
30. Badovinac, V. P., K. A. Messingham, A. Jabbari, J. S. Haring, and J. T. Harty.
2005. Accelerated CD8⫹ T-cell memory and prime-boost response after dendritic-cell vaccination. Nat. Med. 11: 748 –756.
31. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two
subsets of memory T lymphocytes with distinct homing potentials and effector
functions. Nature 401: 708 –712.
32. Lappin, M. B., J. M. Weiss, V. Delattre, B. Mai, H. Dittmar, C. Maier, K. Manke,
S. Grabbe, S. Martin, and J. C. Simon. 1999. Analysis of mouse dendritic cell
migration in vivo upon subcutaneous and intravenous injection. Immunity 98:
181–188.
33. Eggert, A. A., M. W. Schreurs, O. C. Boerman, W. J. Oyen, A. J. de Boer,
C. J. Punt, C. G. Figdor, and G. J. Adema. 1999. Biodistribution and vaccine
efficiency of murine dendritic cells are dependent on the route of administration.
Cancer Res. 59: 3340 –3345.
34. Morse, M. A., R. E. Coleman, G. Akabani, N. Niehaus, D. Coleman, and
H. K. Lyerly. 1999. Migration of human dendritic cells after injection in patients
with metastatic malignancies. Cancer Res. 59: 56 –58.
35. Okada, N., M. Tsujino, Y. Hagiwara, A. Tada, Y. Tamura, K. Mori, T. Saito,
S. Nakagawa, T. Mayumi, T. Fujita, and A. Yamamoto. 2001. Administration
route-dependent vaccine efficiency of murine dendritic cells pulsed with antigens.
Br. J. Cancer 84: 1564 –1570.
36. Olasz, E. B., L. Lang, J. Seidel, M. V. Green, W. C. Eckelman, and S. I. Katz.
2002. Fluorine-18 labeled mouse bone marrow-derived dendritic cells can be
detected in vivo by high resolution projection imaging. J. Immunol. Methods 260:
137–148.
CD8 T CELL REDISTRIBUTION TO LYMPH NODES