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*
Trudeau Institute, Saranac Lake, NY 12983; and
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2 or the type 2 cytokines IL-4, IL-5,
and IL-10, respectively. To study whether the polarized cytokine
phenotype of the effectors is stable, we generated highly cytotoxic
hemagglutinin (HA) peptide-specific CD8 Tc1 and Tc2 (cytotoxic CD8 T
cells producing type 1 or type 2 cytokines) effectors from Clone-4
TCR-transgenic mice, which were adoptively transferred into syngeneic
adult thymectomized irradiated and bone marrow-reconstituted
recipients. The highly activated blast-size, CD25+
Tc1 and Tc2 effectors gave rise to homogeneous resting
CD25-CD44highLy6Chigh Ag-specific
populations, which persisted for at least 13 wk after adoptive
transfer. These memory CD8 T cells, recovered 13 wk after transfer of
Tc1 or Tc2 effectors, still produced either the type 1 or type 2
cytokines, i.e., IFN-, or IL-4 and IL-5, respectively, upon
restimulation with APCs loaded with the HA peptide, but not in the
absence of Ag. The amounts of IL-2 detected in the supernatants of Tc1
and Tc2 memory populations were comparable to that in memory CD4 cells,
and both Tc1 and Tc2 memory cells became cytotoxic upon restimulation.
Thus, cytokine-polarized CD8 memory T cells are a source of a variety
of cytokines, which were classically considered helper cytokines,
opening new perspectives on their function as regulatory cells in an
immune response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(1). Recently, it has become clear that CD8 T
cells are capable of producing a variety of different cytokines in
addition to IFN-, contributing to their effector function (2, 3, 4).
Depending on the cytokines present during primary stimulation, CD8 T
cells differentiate into type 1 and type 2 CD8 T cells producing
IFN- and IL-2 or IL-4, IL-5, IL-6, and IL-10, respectively (reviewed
in Refs. 5 and 6). To date, the ability to produce a polarized pattern
of cytokines has been demonstrated for primary highly
activated CD8 T cell effectors generated from naive transgenic CD8 T
cells responding to their specific Ag in the presence of the polarizing
cytokines (7) and for CD8 T cell clones (8). Moreover, type 1 and type
2 CD8 T cell clones could be generated in vitro from rat T cells (9)
and from human neonatal cord blood T cells (10). In several studies,
including our own, both Tc13 and Tc2 effector CD8 T
cell populations were cytolytic (8, 11, 12), and we use the Tc1 and Tc2
designation throughout this study. However, under certain circumstances
a loss of cytotoxicity with CD8 cells cultured under type 2 inducing
conditions was observed (13, 14).
There is substantial evidence that CD8 T cells secreting type 1 and
type 2 cytokines also exist in vivo and that the polarized pattern of
secreted cytokines might have great relevance to immune responses
against infectious agents, determining whether an immune response will
be successful or deleterious (15). IL-4- and IL-5-secreting CD8 T cells
have been isolated from lesions from patients with lepromatous leprosy
(2) and from HIV-infected individuals with a Job-like syndrome (16).
Furthermore, lymphocytic choriomeningitis virus (LCMV)-specific CD8 T
cells secreting IL-5 have been shown to be associated with airway
eosinophilia (17). IL-5- or IFN--producing CD8 T cells were found in
murine gut-associated tissues (18). Upon adoptive transfer, both in
vitro generated Tc1 and Tc2 cells induced similar delayed-type
hypersensitivity reactions (19). However, in the model of graft-vs-host
disease, adoptive transfer of type 2 CD8 T cells resulted in less
severe disease (12, 20).
During an immune response, naive CD8 T cells or naive CD4 cells
are primed to Ags presented in the context of MHC class 1 molecules or
MHC class 2 molecules, respectively, expand, and acquire effector
functions. After the acute immune response has subsided, an increased
frequency of Ag-specific memory T cells is maintained for years after
the initial priming, which protect against reinfection (21, 22, 23). Naive
CD8 T cells and CD4 T cells represent a homogeneous small resting T
cell population with low CD44 expression that do not produce any
cytokines other than low levels of IFN- or IL-2, respectively, but
can be efficiently primed with Ag and cytokines to become primary
effectors (7). The cytokines present during the initial priming of T
cells determine whether a type 1 or a type 2 effector T cell will
develop (7, 8). For CD4 T cells it has been established that the
cytokines present during the initial priming of the naive cell
population influence the quality of cytokines produced much later by
persisting CD4 memory cells (24). This is true whether transgenic T
cells from wild-type animals or RAG-2-/- mice were
studied (S. L. Swain, manuscript in preparation). The persistence
of memory T cells was observed in adoptive transfer models using
athymic irradiated and bone marrow-reconstituted (ATXBM) animals (24)
or normal mice (25) as adoptive hosts. Whether memory CD8 T cells can
be a source of a variety of different cytokines has not yet been
determined. Furthermore, the nature of the CD8 T cell memory
compartment is complex, and the existence of other subsets of CD8
memory cells defined by the expression of surface molecules, CTL
activity, and cell cycle status has been proposed (26, 27, 28).
In this study, we tested whether adoptive transfer of in vitro generated CD8 effectors producing Tc1 and Tc2 cytokines can give rise to memory populations that maintain the ability to produce the same polarized pattern of cytokines. The CD8 memory cells were defined operationally as long-lived, Ag-reactive, resting CD44high, Ly6Chigh T cells derived from cells in the original effector T cell population. The experimental model used the adoptive transfer of polarized Ag-specific CD8 effectors from the Clone-4 TCR-transgenic mice (29) into ATXBM animals, as described previously for the generation of memory CD4 T cells (24), and allowed us to visualize the nature of the CD8 memory cells in the absence of Ag-specific CD4 cells and in the apparent absence of Ag.
Here we report that resting CD44high, Ly6Chigh, CD25- CD8 T cell populations were established in the recipients of in vitro generated CD8 T cell effectors producing type 1 and type 2 cytokines and could be recovered from the adoptive hosts as long as 13 wk after transfer. The recovered CD8 T cells produced the same polarized pattern of cytokines as the adoptively transferred CD8 Tc1 and Tc2 effectors, demonstrating that polarized CD8 memory cells persist in vivo. Both populations became cytolytic upon restimulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice were purchased from the Animal Breeding Facility at the
Trudeau Institute (Saranac Lake, NY). Clone-4 vß8.2/v
10
TCR-transgenic mice were provided by Dr. Linda Sherman (The Scripps
Research Institute, La Jolla, CA) (29). Clone-4 TCR-transgenic mice
bear the - and ß-chains of the Clone-4 CTL specific for the
transmembrane peptide, residues 518 to 528 (IYSTVASSL) of hemagglutinin
(HA) 2 on H-2Kd. Clone-4 TCR-transgenic mice were
backcrossed for eight generations with B10.D2 mice. HNT TCR-transgenic
mice carrying the TCR specific for the influenza HA peptide amino acids
126 to 138 (HNTNGVTAACS) presented in the context of I-Ad
were provided by Dr. David Lo (The Scripps Research Institute)
(30).
Cell preparations
CD8 T cells were isolated from the spleen and lymph nodes of Clone-4 TCR transgenic mice and enriched by passing them through nylon wool and treating them with anti-CD4 (RL172.4), anti-heat-stable Ag (J11D), and anti-class II MHC (D3.137, M5114, CA4) mAbs and complement. Splenic APCs were enriched from B10.D2 mice by T cell depletion using anti-Thy1.2 (HO13.14 and F7D5), anti-CD4 (RL172.4), and anti-CD8 (3.155) mAbs. Small resting CD8 T cells were harvested from the bottom interphase of a four-layer Percoll gradient (Sigma, St. Louis, MO). The freshly isolated T cell populations were 90 to 95% CD8+vß8+ T cells.
Effector generation
Cells were cultured in RPMI 1640 (Irvine Scientific, Santa Ana,
CA) supplemented with penicillin, streptomycin, glutamine, 2-ME, HEPES,
and 10% FCS (HyClone, Logan, UT). T-depleted splenic APCs were loaded
with the HA peptide (11 µM) at 37°C for 30 min, treated with
mitomycin C (50 µg/ml; Sigma) at 37°C for 40 min, and washed three
times before use. For effector generation, CD8 T cells from the Clone-4
transgenic mice (2 x 105 cells/ml) were
stimulated with HA peptide-loaded APCs (6 x 105
cells/ml) in the presence of IL-2 (20 U/ml; supernatant from the
X63Ag.IL-2 murine cell line), IL-12 (9.2 U/ml; provided by Dr. Stanley
Wolf, Genetics Institute, Cambridge, MA), and anti-IL-4 mAb (10
µg/ml; 11B11) for Tc1 cultures and in the presence of IL-2 (20 U/ml),
IL-4 (200 U/ml; X63.Ag.IL-4 supernatant), and anti-IFN- mAb
(XMG1.2; 20 µg/ml) for Tc2 cultures. Th1 and Th2 effectors from HNT
TCR-transgenic mice were prepared under similar conditions. After 4
days, in vitro culture effectors were washed, analyzed, and
transferred. On day 4 of culture, effectors were 95 to 99%
CD8+vß8+ cells.
Adoptive transfer and preparation of recovered cells
Sex-matched B10.D2 mice were thymectomized at 5 wk of age, 2 to
4 wk later irradiated with 8.5 Gy, and reconstituted with
107 syngeneic T-depleted bone marrow cells plus
107 CD8 effector cells i.v. within 8 h. The adoptively
transferred mice were sacrificed at the indicated time points, and
cells were prepared from spleens and easily accessible lymph nodes,
including the mesenteric lymph nodes. Total live cell counts were
determined by excluding dead cells by trypan blue staining. In Figure 3
, the percentages of the total that were
CD8+vß8+ were determined by FACS analysis and
the absolute number of CD8+vß8+ cells was
calculated. Memory cell populations were enriched by passing them
through nylon wool and treating them with anti-CD4 (RL172.4),
anti-heat-stable Ag (J11D), and anti-class II MHC (D3.137,
M5114, CA4) mAbs and complement. Eighty-five to ninety percent of the
recovered enriched memory cell population was
CD8+vß8+ cells. For phenotypical and
functional analysis, typically a pool of enriched CD8 T cells from at
least two or three mice was used.
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The following mAbs were used for immunofluorescent staining:
Cy-chrome anti-CD8 (PharMingen, San Diego, CA), anti-vß8 PE
(PharMingen; clone MR5–2), FITC anti-CD62L (PharMingen; clone
Mel-14), FITC anti-CD44 (PharMingen; clone IM7), FITC
anti-CD45RB (PharMingen; clone 23G2), FITC anti-CD25
(PharMingen; IL-2R, -chain, clone 3C7), FITC anti-Ly6C
(PharMingen; clone AL-21), FITC anti-LFA-1 (CD11a, PharMingen;
clone 2D7). After staining with the appropriate mAbs, FACS analysis was
conducted on a FACScan (Becton Dickinson, San Jose, CA) using the
CellQuest software. All plots shown were gated on the live cell
population.
Analysis of cytokine production
Enriched CD8 T cells (2 x 105 cells/ml)
were stimulated with mitomycin-treated P815 cells (1.2 x
106/ml) loaded with the HA peptide or unloaded. For
cytotoxicity assays, CD8 T cells were expanded with plate-bound
anti-CD3 mAb (2C11; 10 µg/ml), anti-CD28
(17N51.1.1.3.7.3.2.1; 10 µg/ml), and IL-2 (20 U/ml). Culture
supernatants were harvested at the indicated time points. IL-2 was
detected by measuring the proliferation of the NK3 cell line, which
responds to both IL-2 and IL-4. The anti-IL4 mAb 11B11 was added to
the assay to block IL-4-induced proliferation. The lower limit of
detection of assay detection of IL-2 production was 5 U/ml. IFN- and
IL-5 were measured by specific ELISAs using the anti-IFN- mAbs
R46A2 and XMG1.2 and the anti-IL-5 mAbs TRFK4 and TRFK5, as
previously described (31, 32). The IL-4 ELISA employs the 11B11 mAb for
capture and the biotinylated BVD6-24G2 (PharMingen) for detection. For
measuring IL-10, the anti-IL-10 mAb JES5-2A5 (PharMingen) was used
for capture, and the biotinylated mAb SXC-1 (PharMingen) was used for
detection. One unit of IL-2 corresponds to approximately 14 pg, 1 U of
IL-4 is about 0.7 pg, 1 U of IL-5 is approximately 150 pg, 1 U of
IFN- is about 100 pg, and 1 U of IL-10 is approximately 50
pg.
Intracellular cytokine staining
For assessment of cytokine production by intracellular staining,
polarized effector populations were restimulated at 2 x
105 T cells/ml with 1 x 106
Ag-pulsed, mitomycin C-treated APCs. After 4.5 h, 3 µM monensin
(Calbiochem, La Jolla, CA) was added, and restimulation was continued
for another 10 h. The staining procedure was performed as
previously described (33, 34). Briefly, cells were harvested from
monensin-treated restimulation cultures, washed with PBS, treated with
normal mouse serum to block Fc receptor interactions, and stained for
CD4 or CD8 expression using Cy-chrome-conjugated Abs. After washing,
cells were resuspended in PBS and fixed for 20 min at room temperature
by addition of an equal volume of phosphate-buffered formalin (Fisher,
Pittsburgh, PA). Following two washes in PBS, cells were resuspended in
a saponin-containing permeabilization buffer, incubated at room
temperature for 10 min, pelleted, and resuspended in 100 µl of
permeablization buffer. Five microliters of normal rat serum was added
to block nonspecific interactions for 5 to 10 min at room temperature,
then anti-cytokine (20 µg/ml anti-IFN--FITC and 20 µg/ml
anti-IL-4-PE; PharMingen) or isotype-matched control Abs were
added, and samples were incubated for 20 min at room temperature. Cells
were washed twice in permeabilization buffer and once in PBS, and
analyzed on a FACScan immediately.
Cytotoxicity assays
P815 cells, which were used as targets, were loaded with the HA peptide (11 µM) for 30 min at 37°C. Target cells were labeled by incubating 1 x 106 cells in 400 µl of RPMI medium containing 1% FCS with 3.7 mBq of 51Cr (sp. act., 1.85 TBq/g; New England Nuclear, Boston, MA) for 1 h at 37°C. Labeled targets were washed three times before use. CD8 T cells were set up at the indicated ratios with the labeled targets (104 targets/well) at 37°C, supernatants were collected after 4 h, and radioactivity was detected by gamma counting. Means and SDs of duplicate cultures are shown. The percentage of cytotoxicity was calculated using the formula: 100 x (cpm experimental - cpm spontaneous)/(cpm total - cpm spontaneous).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Naive CD8 T cells were isolated from the Clone-4 TCR-transgenic
mice (29) carrying the v10/vß8.2 TCR specific for a hydrophobic
peptide sequence (amino acids 518–528, IYSTVASSL) from the
transmembrane region of the HA2 molecule from influenza virus. In all
experiments, purified small CD8 T cells were isolated from the bottom
interface of a four-layer Percoll gradient. CD8 T cell effectors
producing type 1 and type 2 cytokines were generated by culturing
transgenic CD8 T cells with HA peptide-loaded, mitomycin-treated,
syngeneic, T-depleted splenocytes in the presence of IL-2, IL-12, and
anti-IL-4 mAb or IL-2, IL-4, and anti-IFN- mAb,
respectively, for 4 days (7). A representative cytokine profile of the
in vitro generated CD8 effector population is shown in Figure 1A. Tc1 effectors produce
IFN-, relatively small amounts of IL-2 (80 U), little IL-10, and no
IL-4 or IL-5 upon stimulation with mitomycin-treated HA peptide-loaded
P815 cells used as APCs (Fig. 1A). No cytokine
production was observed with APCs in the absence of HA peptide (data
not shown), indicating that the response was highly Ag specific. In
contrast, Tc2 effectors produced IL-4, IL-5, IL-10, and substantially
lower amounts of IFN- compared with Tc1 effector cells.
Intracellular cytokine staining revealed that IL-4- and
IFN--containing cells were from separate populations, suggesting
that IFN- production by the Tc2 population came from a contamination
with Tc1 cells (Fig. 1B). However, when Th2 CD4 T
cell effectors generated from HNT TCR-transgenic mice under similar
conditions were analyzed, no intracellular IFN- was detected. Th2
effectors also contained more intracellular IL-4 (12% of cells
positive for intracellular IL-4) compared with Tc2 effectors (5% of
cells positive for intracellular IL-4), which corresponds to the lower
IL-4 protein levels detected in specific ELISAs with Tc2 effectors
(Fig. 1A). In this context, it is important to
emphasize that in our study the polarized primary effector populations
were generated upon one single stimulation with Ag in the presence of
cytokines rather than exposing the cells to repetitive stimulation with
Ag in the continuous presence of the polarizing cytokines as done
routinely with T cell lines and clones, which often leads to a very
clear-cut polarization pattern. However, IFN- production of CD8
cells cultured under type 2 inducing conditions has been observed
previously (12). Both Tc1 and Tc2 effectors were highly cytotoxic to
the HA peptide-loaded P815 cells used as targets but not to targets
without peptide (Fig. 1C). We found no evidence for a
loss in cytotoxicity in the Tc2 cell population as proposed by Erard et
al. (14). In contrast, in our hands the Tc2 population was usually
slightly more cytotoxic than the Tc1 population, ruling out the
possibility that the observed cytotoxicity of the effectors generated
under type 2 inducing conditions was merely due to the contaminating
Tc1 population. On day 4 of stimulation both Tc1 and Tc2 populations
were highly activated blast-size cells with a high forward scatter,
expressing high levels of activation markers, and all cells expressed
CD8 and the transgene vß8 (see Fig. 4, A and B,
and data not shown). No persisting mitomycin-treated APCs were detected
on day 4 by FACS analysis.
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Four days after primary stimulation, in vitro generated Tc1 and
Tc2 effectors were transferred into adoptive hosts. Hosts were adult
thymectomized to ensure that no new host T cells could develop, then
were irradiated with 8.5 Gy and reconstituted on the same day with
T-depleted syngeneic bone marrow together with the in vitro generated
CD8 Tc1 and Tc2 effector populations (24). Animals reconstituted with
bone marrow alone served as controls to ensure that all recovered T
cells were of donor origin. Transgene-positive CD8 T cells were
detected from pooled spleens and lymph nodes 3, 9, and 13 wk after
transfer (Fig. 2). The absolute cell
numbers of recovered CD8+, vß8+ cells
from the spleens and lymph nodes of the adoptive hosts after different
times are shown in Figure 3
. The number
of cells recovered from the tissue samples at 3 wk was at 55% of the
cells injected and declined approximately twofold in the subsequent 10
wk. When spleens and lymph nodes were examined separately, all cell
preparations contained transgene-positive CD8 T cells (data not shown).
CD8 transgene-positive T cells could be detected as long as 20 wk after
adoptive transfer (data not shown). Neither CD8 nor CD4 cells were
found in the animals reconstituted with bone marrow only 3 wk after
adoptive transfer, indicating the completeness of thymectomy. However,
at later time points a few CD4 or CD8 cells (<1.5%), most of which
did not carry the transgene, were detected, but these did not respond
to Ag (data not shown). Together, these data indicate that both Tc1 and
Tc2 effectors persist after adoptive transfer for at least 13 wk
despite the apparent absence of their specific Ag or CD4 T cells in the
adoptive host.
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Naive CD8 T cells represent a homogeneous small resting T cell
population with a low forward scatter profile (Fig. 4A). A similar small
resting phenotype was found with memory cells recovered from the
adoptive hosts at 3 to 13 wk after adoptive transfer. All CD8 effector
cells have a high forward scatter, indicating their activated
blast-size stage. In general, with regard to all surface markers
tested, no significant difference in expression patterns was found
between 4-day Tc1 and Tc2 effectors or Tc1 and Tc2 memory cells,
indicating that the phenotype of Tc1 effector or memory cells, which is
shown in Figure 4B, is representative of both populations.
No obvious phenotypic changes were observed in the recovered T cell
populations between 3 and 20 wk of adoptive transfer; thus, the
phenotype shown at 12 wk is representative of those at all time points
tested. Stainings of the CD8 memory populations before enrichment by
nylon wool, mAbs, and complement were similar to staining after CD8 T
cell enrichment, indicating that we did not lose an activated memory
population during the purification process (data not shown). Naive CD8
T cells were CD44low, CD62Lhigh, and
CD45RBhigh, a phenotype that had been previously associated
with a naive CD8 T cell phenotype (7). Effector cells, generated after
4 days of in vitro culture, up-regulated CD44 and down-regulated
CD45RB. CD44 expression was maintained at high levels on memory T
cells, which were recovered 12 wk after adoptive transfer, whereas
CD45RB was up-regulated to levels similar to those in naive cells. With
regard to the expression of CD62L (MEL-14), effectors were negative,
whereas most of the memory cells expressed CD62L at levels comparable
to those in naive CD8 T cells. However, a small subset of memory cells
(10%) did not express CD62L. Naive and memory CD8 T cells stained
negative for CD25, which was expressed at high levels on effectors.
LFA-1 was expressed at lower levels on naive cells than on effectors
and memory cells.
An interesting expression pattern on naive vs effector vs memory cells was found with the Ly6C mAb. Naive cells could be divided almost equally into Ly6C-/Ly6C+ subsets, whereas both Tc1 and Tc2 effectors had down-regulated Ly6C expression. Both Tc1 and Tc2 memory cells stained very brightly with the Ly6C mAb.
Thus, the persisting resting CD8 population recovered from the adoptive hosts is characterized by a memory phenotype most clearly defined by the expression of a low level of CD25 and high levels of CD44 and Ly6C, which is distinct from either a naive or an effector phenotype.
Cytokine production of the recovered CD8 T cell populations
To assess functional memory with regard to polarized cytokine
production of the recovered cell populations, CD8 cells from adoptive
hosts that had received Tc1 and Tc2 effector populations 9 wk (Fig. 5, upper panel) or 13
wk (Fig. 5, lower panel) earlier were enriched for
CD8 T cells and restimulated with mitomycin-treated APCs pulsed with
the HA peptide or unpulsed (Fig. 5). In parallel, cytokine production
of freshly isolated naive CD8 T cells was determined under similar
conditions. Naive CD8 T cells made comparable low amounts of IFN-
but no other cytokines upon stimulation with their specific Ag.
However, high levels of a variety of cytokines were found in the
supernatants of the recovered memory CD8 T cell populations upon
restimulation with HA peptide-loaded APCs. No cytokine production was
detected when CD8 T cells were stimulated in the absence of the HA
peptide (data not shown). CD8 T cells recovered from hosts that had
received a Tc1 effector population 9 or 13 wk previously produced high
level of IFN- but no IL-4 or IL-5 and little IL-10. In contrast,
IL-4, IL-5, and IL-10 and slightly lower levels of IFN- compared
with those in Tc1 memory cells could be detected in the supernatants
from restimulated CD8 T cells recovered from hosts that had been
injected with Tc2 effectors. Supernatants of both restimulated Tc1 and
Tc2 memory T cells contained considerable levels of IL-2 (
1200 U at
13 wk after transfer). At this point we cannot rule out that the IL-2
production seen with the Tc2 population was due to the contaminating
Tc1 population. However, when kinetics of IL-2 production by Tc1 and
Tc2 memory cells were analyzed, the highest IL-2 production was
observed at 24 h and dropped within the next 48 h. At all
time points the same amounts of IL-2 were detected in the supernatants
of Tc1 and Tc2 memory cells, indicating that at least some IL-2
production might be uncoupled from the IFN- production.
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Recovered CD8 T cells producing type 1 and type 2 cytokines develop into specific CTLs in vitro
Next, we investigated functional memory of our recovered cell
populations as defined by their ability to kill HA peptide-pulsed
targets. Purified CD8 T cells taken straight from the adoptive hosts
displayed low levels of cytotoxicity to P815 cells regardless of
whether the targets were loaded with HA peptide or not loaded in a 4-h
51Cr release assay at a 9:1 E:T cell ratio (Fig. 6A). However, after
expansion with plate-bound anti-CD3 and soluble anti-CD28 mAbs
in the presence of IL-2 for 3 days, highly cytotoxic secondary
effectors developed, which efficiently killed HA peptide-loaded
specific targets but not unspecific targets (Fig. 6B), indicating that the recovered cell populations
gave rise to effector populations with highly specific cytotoxic
activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here, we report that in vitro polarized primary CD8 T cell
effectors producing type 1 and type 2 cytokines persisted up to 13 wk
in adoptive hosts and gave rise to homogeneous resting
CD44high, Ly6Chigh, CD25- CD8
T cell populations (
Figs. 2–4). The use of ATXBM animals as recipients
allowed us to follow the transferred cells in recipients otherwise
devoid of T cells and showed that the cells could persist in the
absence of CD4 T cells. The resting CD8 T cell populations recovered 9
or 13 wk after adoptive transfer of Tc1 or Tc2 effectors still produced
the type 1 and type 2 cytokines, i.e., IFN- or IL-4 and IL-5,
respectively, upon restimulation with specific Ag (Fig. 5). The amounts
of IL-2 detected in the supernatants of restimulated Tc1 and Tc2 memory
cells were comparable to the levels in CD4 memory cells (24). Naive CD8
T cells did not produce any cytokines other than low levels of IFN-
(up to 50 times less than the levels of IFN- produced by memory
cells under the same experimental conditions). Thus, the cytokines
produced by the memory population differed not only qualitatively but
also quantitatively from cytokines produced by freshly isolated naive
CD8 T cells. The production of cytokines and cytolytic activity was Ag
specific, making it likely that the memory cells were derived from
transgene (vß8.2+/v10+)-positive cells and
not from cells using only endogenous receptor chains. Both Tc1 and Tc2
memory cells could be restimulated to exert specific cytotoxicity in
vitro (Fig. 6).
The adoptive transfer of 107 Tc1 or Tc2 effectors
resulted in a considerable number of recovered CD8+
vß8+ cells 3, 9, and 13 wk after adoptive transfer (Figs. 2 and 3). However, the absolute number of recovered CD8 T cells was
always considerably lower than the original donor T cell input (Fig. 3). To us this is not surprising. First, the only organs from which we
harvested cells from the adoptive hosts were spleens and lymph nodes.
Thus, we sampled only a fraction of total lymphoid tissue (35). Second,
homeostatic regulation of the size of the peripheral T cell
compartment, as proposed by Rocha et al. (36), may control the number
of T cells in the peripheral T cell compartment, and the number of
recovered cells might remain constant. Indeed, the relative number of
CD8 T cells recovered from whole spleens and lymph nodes (9–13% of
the whole recovered cell population at 9 wk after adoptive transfer;
Fig. 2) corresponded well to CD8 T cell numbers recovered from the same
organs from normal B10.D2 mice (7%) (data not shown). In contrast,
using a similar approach to study CD4 memory generation (24), expansion
of the adoptively transferred cell population, rather than cell loss,
was observed. This finding might reflect a larger expansion potential
for CD4 T cells compared with CD8 T cells, a phenomenon that has been
previously observed (36). Third, it is likely that not all effectors
have the ability to give rise to memory cells, and thus only a small
proportion of effectors meeting these criteria might survive and revert
to the resting stage (37).
We can only speculate about which factors are responsible for the
maintenance of memory cells in our experimental system.
Exposure to depots of Ag has been shown to be necessary for
functional memory in some cases (38, 39) but not in others (25, 40, 41, 42, 43). In our study we believe that there was little chance of
carry-over of Ag into the adoptive host, because the mitomycin-treated
T-depleted splenocytes used as APCs did not survive the primary in
vitro culture. The only potential source of Ag would be peptide
exchange from the preloaded APCs to MHC class 1 molecules on CD8 cells,
which would be expected to result in killing and elimination rather
than survival of HA peptide-carrying CD8 T cells in culture. It has
been proposed that exposure to cross-reactive Ags is sufficient for the
maintenance of long term memory. Our finding that cytokine production
and cytotoxicity of the recovered memory population were highly Ag
specific indicates that the recovered cell population bears the
transgenes vß8 and v10. Whether additional - or ß-chains
(44, 45, 46) were expressed is unknown at this point. However, there is
evidence that CD4 memory cells generated in a similar experimental
set-up from the AND TCR transgenic mice crossed to the
RAG-2-/- background did not depend on the presence of
endogenous receptors for survival (S. L. Swain, manuscript in
preparation). In this study we also did not address the question of
whether cytokines such as type 1 IFN (47) are responsible for the
maintenance of our memory population, and experiments studying memory
generation in cytokine knockout animals are under way.
All vß8+CD8+ T cells recovered from the adoptive hosts are small resting T cells with a low forward scatter, which do not express CD25. This resting memory phenotype has been seen in LCMV immune mice (26) and in several previous studies (25, 41). In addition, a population of blast-size, cycling memory CD8 T cells expressing CD62L and CD25 (26) and containing precursor CTLs has been described in LCMV immune mice. The blast-size memory population in LCMV-immune mice was also shown to mediate cytolysis without in vitro or in vivo restimulation using highly sensitive targets (RMA-S cells) (48) or EL-4 cells (27) as targets. In none of our experiments has such an effector-like, blast-size, cytolytically active memory population been observed, although with regard to the cytotoxicity we cannot rule out that our assays using P815 cells as targets did not reach the required sensitivity. However, our experimental model differed substantially from those of studies conducted in viral systems. First, in LCMV-immune mice the levels of persisting Ag are difficult to determine, whereas our study was conducted in the apparent absence of Ag. Second, cytokines released by Ag-specific CD4 T cells, which were absent in our study, might have influenced the generation and maintenance of the effector-like memory subpopulation. It will be interesting to determine whether the addition of Ag-specific CD4 cells or Ag might give rise to this effector-like CD8 memory population in our system.
With regard to the surface phenotype, the recovered cell population
differed substantially from a naive cell population or an effector cell
population (Fig. 4). The most striking differences defining a resting
naive and a resting memory phenotype were seen with two surface
markers: CD44 and Ly6C. The use of CD44 as a memory marker is widely
established (49, 50), although it does not work in some mouse strains
tested (51). Naive cells are CD44low, effectors become
CD44high, and CD44 expression stays high on memory cells.
Ly6C, however, distinguishes very clearly among the naive, effector,
and memory phenotypes. In the B10.D2 mouse strain, naive cells can be
separated almost equally into Ly6C+ and Ly6C-
subsets. Effectors stain negative, and memory cells stain very brightly
with the Ly6C mAb. Thus, an almost inverse relationship between
activation status and Ly6C expression is present. It has been reported
that within the resting CD8 T cell population, CD44high
cells were found within the Ly6C+ subset in vivo (52). In
our study naive CD8 T cells from transgenic mice homogeneously
expressed low levels of CD44 together with high or low Ly6C expression,
making it unlikely that only Ag-experienced memory cells reside in the
Ly6C+ subset. High Ly6C expression on CD8 memory cells has
been observed in vivo after viral infection (47), peptide immunization
(53), or anti-CD3 treatment (54). Thus, the unidentified ligand of
Ly6C might be critically involved in the generation and function of CD8
memory cells. Expression of the high and the low isoforms of CD45RB is
frequently used to distinguish CD4 memory cells from naive cells in
mice (24) and humans (55), a view that had been challenged by recent
findings that interconversion of the isoforms occurs in vivo in rats
and humans (56, 57, 58). We found high expression of CD45RB on naive cells,
down-regulation on effectors, and subsequent up-regulation on memory
cells. This argues against CD45RB being a good memory marker for CD8 T
cells, which is in concordance with other previous reports (27, 59).
Our data demonstrate functional memory of the recovered cell
populations with two readouts: polarized cytokine production and
specific cytotoxicity. The fact that our recovered CD8 memory cell
population still produced the type 1 or type 2 cytokines, i.e., IFN-
or IL-4 and IL-5, respectively (Fig. 5), adds a new quality to CD8
memory cells in addition to their potential to give rise to specific
CTLs. We show that Tc1 memory cells make far higher levels of IFN-
(up to 50 times more) than naive cells. This level of IFN- produced
by memory cells is also higher than that made by Tc1 effectors cells.
In this context, Sad et al. (60) demonstrated that the capacity of CD8
effector cells to produce high levels of cytokines was limited by their
ability to rapidly kill APCs presenting Ag, which prevented continued
interaction between the CD8 T cells and the APCs. In our study, the CD8
Tc1 and Tc2 memory cells were not cytolytically active unless they were
restimulated (Fig. 6), which could explain the strikingly higher levels
of IFN- and IL-2 produced by CD8 memory cells compared with CD8
effectors. In our experiments naive CD8 T cells did not produce any
detectable IL-2, supernatants of restimulated Tc1 and Tc2 populations
contained amounts of IL-2 comparable to that produced by memory CD4
cells (24). These data suggest that most naive CD8 T cells are
dependent on CD4 T cells for their supply of IL-2, whereas memory CD8 T
cells, at least under our experimental conditions, are a good source of
IL-2, which might lead to CD4 helper cell-independent memory
responses.
The demonstration that the ability to produce a polarized pattern
of cytokines at the effector stage was maintained after the effectors
had reverted to a resting memory stage reemphasizes the importance of
CD8 as a source of a variety of cytokines other than IFN-. Up until
now, the stability of the pattern of cytokines produced by CD8 T cells
has only been shown at a clonal level (8), where repeated stimulation
with Ag resulted in a chronically stimulated T cell population selected
for their survival potential in vitro rather than in a population of
defined differentiation stages. In fact, the cytokines present during
initial priming of a naive CD8 T cell population programmed the quality
of cytokines produced by the same T cell population 13 wk later upon a
second encounter of Ag. This is of particular interest because subsets
of CD8 T cells have been identified in humans as well as in mice during
various infections (15). Specifically in situations in which the
cytotoxicity and IFN- production by CD8 T cells are protective, the
switch to IL-4 and IL-5 production might allow a pathogen to escape
elimination and vice versa. The type 2 memory cells recovered from the
adoptive hosts in our study represent a complete new category CD8
memory cells: they produce helper cytokines, they do not kill, but they
can be restimulated for specific cytotoxicity in vitro. It will be
highly interesting to establish the protective value of this cell
population in vivo against infectious agents.
Our data have considerable application to adoptive immunotherapy using Ag-specific CTLs in anti-viral (61) or anti-tumor therapy. In humans, adoptive immunotherapy with CMV-specific CTLs has been successfully conducted (62). Thus in disease for which the type 2 response might be correlated with the progress of disease, such as in HIV infection (63), adoptive transfer of autologous CTLs producing type 1 cytokines should not only result in effective killing of virus-infected target cells but should also help to establish persisting memory of the "right" subset.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard W. Dutton, Trudeau Institute, Algonquin Avenue, Saranac Lake, NY 12983. E-mail address:
3 Abbreviations used in this paper: Tc1, Tc2, cytotoxic CD8 T cells producing type 1 or type 2 cytokines; LCMV, lymphocytic choriomeningitis virus; ATXBM, athymic irradiated bone marrow reconstituted; HA, hemagglutinin; PE, phycoerythrin; RAG, recombination activating gene; HNT TCT-transgenic mice, mice having the TCR specific for the HNT (HNTNGVTAACS) peptide.
Received for publication December 5, 1997. Accepted for publication February 27, 1998.
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P. Gorak-Stolinska, J.-P. Truman, D. M. Kemeny, and A. Noble Activation-induced cell death of human T-cell subsets is mediated by Fas and granzyme B but is independent of TNF-{alpha} J. Leukoc. Biol., November 1, 2001; 70(5): 756 - 766. [Abstract] [Full Text] [PDF]
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T. Chtanova, R. A. Kemp, A. P. R. Sutherland, F. Ronchese, and C. R. Mackay Gene Microarrays Reveal Extensive Differential Gene Expression in Both CD4+ and CD8+ Type 1 and Type 2 T Cells J. Immunol., September 15, 2001; 167(6): 3057 - 3063. [Abstract] [Full Text] [PDF]
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B.-C. Sheu, R.-H. Lin, H.-C. Lien, H.-N. Ho, S.-M. Hsu, and S.-C. Huang Predominant Th2/Tc2 Polarity of Tumor-Infiltrating Lymphocytes in Human Cervical Cancer J. Immunol., September 1, 2001; 167(5): 2972 - 2978. [Abstract] [Full Text] [PDF]
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 A. Magnan, V. Di Bartolo, A.-M. Mura, C. Boyer, M. Richelme, Y.-L. Lin, A. Roure, A. Gillet, C. Arrieumerlou, O. Acuto, et al. T Cell Development and T Cell Responses in Mice with Mutations Affecting Tyrosines 292 or 315 of the Zap-70 Protein Tyrosine Kinase J. Exp. Med., August 20, 2001; 194(4): 491 - 506. [Abstract] [Full Text] [PDF]
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S. W. Chensue, N. W. Lukacs, T.-Y. Yang, X. Shang, K. A. Frait, S. L. Kunkel, T. Kung, M. T. Wiekowski, J. A. Hedrick, D. N. Cook, et al. Aberrant in Vivo T Helper Type 2 Cell Response and Impaired Eosinophil Recruitment in Cc Chemokine Receptor 8 Knockout Mice J. Exp. Med., March 5, 2001; 193(5): 573 - 584. [Abstract] [Full Text] [PDF]
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R. J. Hogan, E. J. Usherwood, W. Zhong, A. D. Roberts, R. W. Dutton, A. G. Harmsen, and D. L. Woodland Activated Antigen-Specific CD8+ T Cells Persist in the Lungs Following Recovery from Respiratory Virus Infections J. Immunol., February 1, 2001; 166(3): 1813 - 1822. [Abstract] [Full Text] [PDF]
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C. Vizler, N. Bercovici, A. Heurtier, N. Pardigon, K. Goude, K. Bailly, C. Combadiere, and R. S. Liblau Relative Diabetogenic Properties of Islet-Specific Tc1 and Tc2 Cells in Immunocompetent Hosts J. Immunol., December 1, 2000; 165(11): 6314 - 6321. [Abstract] [Full Text] [PDF]
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J. B. Reome, D. S. Johnston, B. K. Helmich, T. M. Morgan, N. Dutton-Swain, and R. W. Dutton The Effects of Prolonged Administration of 5-Bromodeoxyuridine on Cells of the Immune System J. Immunol., October 15, 2000; 165(8): 4226 - 4230. [Abstract] [Full Text] [PDF]
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A. W. Goldrath, L. Y. Bogatzki, and M. J. Bevan Naive T Cells Transiently Acquire a Memory-like Phenotype during Homeostasis-Driven Proliferation J. Exp. Med., August 21, 2000; 192(4): 557 - 564. [Abstract] [Full Text] [PDF]
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M. Kaji, M. Kobayashi, R. B Pollard, and F. Suzuki Influence of type 2 T cell responses on the severity of encephalitis associated with influenza virus infection J. Leukoc. Biol., August 1, 2000; 68(2): 180 - 186. [Abstract] [Full Text]
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V. Ganapathy, T. Gurlo, H. O. Jarstadmarken, and H. von Grafenstein Regulation of TCR-induced IFN-{gamma} release from islet-reactive non-obese diabetic CD8+ T cells by prostaglandin E2 receptor signaling Int. Immunol., June 1, 2000; 12(6): 851 - 860. [Abstract] [Full Text] [PDF]
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H. Ma and J. A. Kapp Antigenic Epitopes Regulate the Phenotype of CD8+ CTL Primed by Exogenous Antigens J. Immunol., June 1, 2000; 164(11): 5698 - 5703. [Abstract] [Full Text] [PDF]
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P. R. Rogers, C. Dubey, and S. L. Swain Qualitative Changes Accompany Memory T Cell Generation: Faster, More Effective Responses at Lower Doses of Antigen J. Immunol., March 1, 2000; 164(5): 2338 - 2346. [Abstract] [Full Text] [PDF]
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M. J. Dobrzanski, J. B. Reome, and R. W. Dutton Type 1 and Type 2 CD8+ Effector T Cell Subpopulations Promote Long-Term Tumor Immunity and Protection to Progressively Growing Tumor J. Immunol., January 15, 2000; 164(2): 916 - 925. [Abstract] [Full Text] [PDF]
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A. Cerwenka, T. M. Morgan, and R. W. Dutton Naive, Effector, and Memory CD8 T Cells in Protection Against Pulmonary Influenza Virus Infection: Homing Properties Rather Than Initial Frequencies Are Crucial J. Immunol., November 15, 1999; 163(10): 5535 - 5543. [Abstract] [Full Text] [PDF]
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J. Schwarze, M. Makela, G. Cieslewicz, A. Dakhama, M. Lahn, T. Ikemura, A. Joetham, and E. W. Gelfand Transfer of the Enhancing Effect of Respiratory Syncytial Virus Infection on Subsequent Allergic Airway Sensitization by T Lymphocytes J. Immunol., November 15, 1999; 163(10): 5729 - 5734. [Abstract] [Full Text] [PDF]
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A. G. Doyle, K. Buttigieg, P. Groves, B. J. Johnson, and A. Kelso The Activated Type 1-Polarized Cd8+ T Cell Population Isolated from an Effector Site Contains Cells with Flexible Cytokine Profiles J. Exp. Med., October 18, 1999; 190(8): 1081 - 1092. [Abstract] [Full Text] [PDF]
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E. S. N. Lamouse-Smith, D. S. Dougall, and S. A. McCarthy Cytokine Requirements for Production of a Novel Anti-CD8-Resistant CTL Population J. Immunol., October 15, 1999; 163(8): 4160 - 4167. [Abstract] [Full Text] [PDF]
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M. Ahmadzadeh, S. F. Hussain, and D. L. Farber Effector CD4 T Cells Are Biochemically Distinct from the Memory Subset: Evidence for Long-Term Persistence of Effectors In Vivo J. Immunol., September 15, 1999; 163(6): 3053 - 3063. [Abstract] [Full Text] [PDF]
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M. Rodolfo, C. Zilocchi, P. Accornero, B. Cappetti, I. Arioli, and M. P. Colombo IL-4-Transduced Tumor Cell Vaccine Induces Immunoregulatory Type 2 CD8 T Lymphocytes That Cure Lung Metastases Upon Adoptive Transfer J. Immunol., August 15, 1999; 163(4): 1923 - 1928. [Abstract] [Full Text] [PDF]
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H. Sepulveda, A. Cerwenka, T. Morgan, and R. W. Dutton CD28, IL-2-Independent Costimulatory Pathways for CD8 T Lymphocyte Activation J. Immunol., August 1, 1999; 163(3): 1133 - 1142. [Abstract] [Full Text] [PDF]
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H. Xie, Y.-C. Lim, F. W. Luscinskas, and A. H. Lichtman Acquisition of Selectin Binding and Peripheral Homing Properties by CD4+ and CD8+ T Cells J. Exp. Med., June 7, 1999; 189(11): 1765 - 1776. [Abstract] [Full Text] [PDF]
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M. J. Dobrzanski, J. B. Reome, and R. W. Dutton Therapeutic Effects of Tumor-Reactive Type 1 and Type 2 CD8+ T Cell Subpopulations in Established Pulmonary Metastases J. Immunol., June 1, 1999; 162(11): 6671 - 6680. [Abstract] [Full Text] [PDF]
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J. Schwarze, G. Cieslewicz, A. Joetham, T. Ikemura, E. Hamelmann, and E. W. Gelfand CD8 T Cells Are Essential in the Development of Respiratory Syncytial Virus-Induced Lung Eosinophilia and Airway Hyperresponsiveness J. Immunol., April 1, 1999; 162(7): 4207 - 4211. [Abstract] [Full Text] [PDF]
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A. Cerwenka, T. M. Morgan, A. G. Harmsen, and R. W. Dutton Migration Kinetics and Final Destination of Type 1 and Type 2 CD8 Effector Cells Predict Protection against Pulmonary Virus Infection J. Exp. Med., January 18, 1999; 189(2): 423 - 434. [Abstract] [Full Text] [PDF]
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F. Erard, J. A. Garcia-Sanz, R. Moriggl, and M.-T. Wild Presence or Absence of TGF-{beta} Determines IL-4-Induced Generation of Type 1 or Type 2 CD8 T Cell Subsets J. Immunol., January 1, 1999; 162(1): 209 - 214. [Abstract] [Full Text] [PDF]
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