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Trudeau Institute, Saranac Lake, NY 12983
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, or resting Tc1 memory cells,
generated from these effectors, were protective. Highly activated
CD62Llow Tc1 effectors accumulated in the lung with rapid
kinetics and most efficiently reduced the pulmonary viral titer early
during infection. The resting CD62Lhigh naive and memory
populations first increased in cell numbers in the draining lymph
nodes. Subsequently, memory cells accumulated more rapidly and to a
greater extent in the lung lavage as compared with naive cells. Thus,
effector cells are most effective against a localized virus infection,
which correlates with their ability to rapidly distribute at the
infected tissue site. The finding that similar numbers of naive
Ag-specific CD8 T cells are not protective supports the view that
qualitative differences between the two resting populations, the naive
and the memory population, may play a major role in their protective
value against disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cellular basis for CD8 T cell-mediated protection against a localized viral infection is only partially known. In particular, the relative effectiveness of CD8 T cell populations during different stages of their differentiation in adoptive transfer models has not been previously defined. In an unprimed animal, the frequencies of cells specific for a defined Ag are very low (7, 8), but very high frequencies of virus-specific cells are generated during infection (5, 9, 10), which in most cases protect against reinfection. High frequencies of naive CD8 T cells present in TCR-transgenic mice specific for the nuclear protein peptide from influenza virus did not clear influenza virus from the lungs when infected with a high viral dose, and a second role for CD8 T cells causing immunopathology was implicated (11). A recent study suggested that qualitative differences rather than quantitative differences were responsible for a more efficient memory CD8 response against a localized tumor (12). Highly activated T effector cells, however, have been shown to undergo rapid activation-induced cell death when restimulated with their specific Ag in vitro (13), and thus might not be thought to be effective in promoting host recovery when adoptively transferred in vivo.
Previous studies addressing the protective value of memory populations were often complicated by the complexity of the experimental models used and the heterogeneity of the memory populations studied. To date, most investigators have determined memory responses in disease models in which primary and secondary infections were conducted in the same hosts. In these studies, several components of the cellular and humoral immune system played together and resulted in a more efficient memory immune response. When the CD8 memory T cell response during lymphocytic choriomeningitis virus (LCMV)3 (14) or influenza virus infection (8) was determined, the primary and secondary infection had to be conducted with different but related virus strains to circumvent strong neutralizing recall Ab responses. In addition, differences in the localization of memory cells presumably dependent on the route of immunization in the primed host might influence the memory response of the primed animal (15), and in most models studied, the impact of locally persisting Ag is largely unknown. The issue of locally persisting Ag is of particular importance, since it was previously suggested that after LCMV infection, only activated cells extravasated efficiently to a peripheral site of infection and provided protection, and continuous Ag exposure was found to be effective in keeping CD8 T cells in an activated stage (16, 17). When CD8 memory cells in LCMV-immune animals were studied, subpopulations of CD8 memory cells were phenotypically and functionally defined (18, 19, 20). In addition to a resting memory cell population, a highly activated memory cell population consisting of blast-like CD25positive cells, which were cytolytic immediately ex vivo of the animal, was described. These subpopulations might differ profoundly in their activation requirements and protective value against disease.
In our study, we addressed the question of how equal numbers of three defined populations, naive CD8 T cells, in vitro generated effector Tc1 CD8 T cells, and resting Tc1 memory CD8 T cells, differed in their protective value against pulmonary influenza virus infection. The CD8 memory cells, generated by the injection of in vitro generated Tc1 effectors into adult thymectomized, irradiated, and bone marrow-reconstituted hosts, were defined operationally as long-lived, Ag-reactive, resting CD44high, CD62high, Ly-6Chigh T cells, as previously described (21). The experimental model utilized the adoptive transfer of Thy-1.2-positive CD8 T cells from the clone-4 TCR-transgenic mice specific for the HA peptide of influenza virus (A/PR/8/34) into congenic Thy-1.1 BALB/c hosts. Ag-specific naive, effector, and memory populations were transferred into unprimed age-matched recipients to minimize differences between different groups of recipients.
We show that depending on their stage of differentiation, identical numbers of CD8 T cells were very different in their protective value against pulmonary influenza virus infection, despite the fact that they bore identical receptors specific for the Ag. The protective value of CD8 T cell populations was correlated with their ability to distribute at the infected tissue site early during infection. Adoptive transfer of either effector or memory cells, but not naive cells, promoted host survival, but only effectors accumulated in the lung with rapid kinetics and reduced the pulmonary virus titer early during infection. Donor cells derived from memory cells entered the lung later than effectors, but slightly earlier than cells derived from naive cells, and subsequently accumulated in the lung lavage in high numbers.
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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. The clone-4 Vß8.2/V
10 TCR-transgenic mice were
kindly provided by Dr. Linda Sherman (The Scripps Research Institute,
La Jolla, CA) (22). T cells from the clone-4
TCR-transgenic mice are Thy-1.2 positive and bear the - and
ß-chains of the clone-4 CTL specific for the transmembrane peptide,
residues 518–528 (IYSTVASSL) of HA2 on H-2Kd.
The clone-4 TCR-transgenic mice were backcrossed for at least 12
generations with BALB/c mice. BALB/c Thy-1.1 mice were a gift from Dr.
Jon Sprent (The Scripps Research Institute).
Cell preparations
Naive CD8 T cells from the spleens and lymph nodes of clone-4 TCR-transgenic mice were enriched by passing through nylon wool and treating with anti-CD4 (RL172.4), and anti-heat-stable Ag (J11D), anti-class II MHC (D3.137, M5114, CA4) mAbs, and complement. Small resting CD8 T cells were harvested from the bottom interface of a four-layer Percoll gradient (Sigma, St. Louis, MO). The freshly isolated T cell populations were 90–95% CD8+Vß8+ T cells and were phenotypically naive, as described (21). For effector generation, CD8 T cells from the clone-4 transgenic mice (2 x 105 cells/ml) were stimulated with HA peptide-loaded APCs (2 x 105 cells/ml) in the presence of IL-2 (100 U/ml, supernatant from the X63Ag.IL-2 murine cell line), IL-12 (9.2 U/ml, kindly provided by Dr. Stanley Wolf, Genetics Institute, Cambridge, MA), and anti-IL-4 mAb (10 µg/ml, 11B11) for Tc1 cultures. T cell-depleted APCs were prepared using anti-Thy-1.2 (HO13.14 and F7D5), anti-CD4 (RL172.4), anti-CD8 (3.155) mAbs, and complement and stimulated with LPS (25 µg/ml) and dextran sulfate (25 µg/ml) for 48 h. APC blasts 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. CD8 T cells were cultured in RPMI 1640 (Irvine Scientific, Santa Ana, CA) supplemented with penicillin, streptomycin, glutamine, 2-ME, HEPES, and 10% FCS (HyClone Laboratories, Logan, UT). On day 4 of culture, effectors were 99% CD8+Vß8+. Tc1 memory cells were generated as described (21): briefly, sex-matched BALB/c mice were thymectomized at 5 wk of age, and 2–4 wk later irradiated with 8.5 Gy and reconstituted with 107 syngeneic T-depleted bone marrow cells plus 107 Tc1 CD8 effector cells i.v. within 8 h. The adoptively transferred mice were sacrificed typically 8–10 wk after transfer, and cells were prepared from spleens and easily accessible lymph nodes. Memory cells were enriched by passing through nylon wool and Ab and complement depletion, as described above, for the preparation of naive cells and were typically 80–90% CD8+Vß8+-positive cells.
Virus infections and cell transfers
The influenza virus preparation (A/PR/8/34) grown in the allantoic cavity of 10-day-old embryonated hens’ eggs was a kind gift of Drs. David Morgan and Linda Sherman (The Scripps Research Institute). Each mouse was inoculated with a 10 LD50 dose of virus (corresponding to a 10-3 dilution of stock virus in 100 µl PBS) intranasally during light halothane (Halocarbon, River Edge, NY) anesthesia. Before adoptive transfer, CD8 cell populations were washed twice in HBSS, and 107 CD8 T cells, if not indicated otherwise, were injected in 0.5 ml PBS via the tail vein within approximately 1 h after intranasal virus infection.
MDCK virus plaque assay
Viral titers of infected lungs were determined using MDCK cell plaque assay modified from the methods described by Lukacher et al. (4). Briefly, at the indicated time points after influenza virus infection, lungs were snap frozen in liquid nitrogen and stored at -70°C until ready for titration. MDCK monolayers were grown in DMEM supplemented with 10% FCS, 0.01 mM nonessential amino acids, 1 mM sodium pyruvate, and PSG (200 IU/ml penicillin, 200 µg/ml streptomycin, and 4 mM glutamine). Ten-fold dilutions of the lung homogenates were prepared in DMEM supplemented with 0.2% BSA, 2 mg/ml NaHCO3, 2 mM HEPES, and PSG. A total of 100 µl of each dilution was added to confluent monolayers of MDCK cells in 12-well plates in duplicates for 1 h at 37°C, 7% CO2. Each well received 1 ml of an agar overlay medium containing DMEM, 0.2% BSA, 2 mg/ml NaHCO3, 2 mM HEPES, PSG, 0.5% agar (Sigma), 0.01% DEAE dextran, and 0.5 mg/ml trypsin. After a 3-day incubation at 37°C, cells were fixed with 0.5 ml Carnoy’s fixative (3:1, methanol:glacial acetic acid) for at least 20 min. The agar overlay was then removed and fixed monolayers were stained by adding a 1/10 dilution of crystal violet prepared in 20% ethanol. The results are presented as PFU/ml = (mean number of plaques/0.1) x (1/dilution factor).
Flow cytometry
The following mAbs were used for immunofluorescent staining:
Cy-chrome anti-CD8 (PharMingen, San Diego, CA), FITC anti-CD62L
(PharMingen; clone Mel-14), FITC anti-CD44 (PharMingen; clone IM7),
FITC anti-CD25 (PharMingen; IL-2R, -chain, clone 3C7), FITC
anti-Ly-6C (PharMingen; clone AL-21), and PE anti-CD90
(Thy-1.2) (PharMingen). After staining with the appropriate mAbs,
FACS analysis was conducted on a FACScan (Becton Dickinson, San Jose,
CA) by using the Cell Quest software.
Tissue sampling
Tissues were sampled by bronchoalveolar lavage performed as follows. Mice were anesthetized and bled out from the axilla. The trachea was then exposed, and a disposable plastic cannula with a syringe attached was then inserted through an incision immediately posterior to the larynx. The respiratory tract was washed out in a reproducible manner using four separate 1-ml aliquots of PBS containing 3 mM EDTA. Subsequently, the lavaged cells were centrifuged and live cell counts were performed by excluding trypan blue-positive dead cells.
Intracellular cytokine staining
For assessment of IFN- cytokine production by intracellular
staining, cell populations from the bronchoalveolar lavage, spleens,
and TBLN (1 x 106 cells/ml) were
restimulated with mitomycin C-treated P815 cells (1 x
106/ml) loaded with the HA peptide (11 µM) or
unloaded. Four hours after the initiation of culture, 10 µM/ml
Brefeldin (Sigma) was added and restimulation was continued for another
10 h. The staining procedure was performed as previously described
(23). Briefly, cells were harvested from Brefeldin-treated
restimulation cultures, washed with PBS, treated with normal mouse
serum to block FcR interactions, and stained with Thy-1.2 PE
(PharMingen) and CD8 Cy-chrome mAbs (PharMingen). After washing, cells
were fixed for 20 min at room temperature with 75 µl 4% freshly
prepared paraformaldehyde (Fisher, Pittsburgh, PA). Following one wash
in PBS, cells were resuspended in a saponin-containing permeabilization
buffer containing FITC-conjugated anti-IFN- mAb (1 µg/ml;
PharMingen) or the respective isotype control, stained for 30 min at
room temperature, washed in PBS, and analyzed immediately on a FACScan.
No positive staining was detected when cells were incubated with the
respective isotype control mAb.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First, we tested whether adoptive transfer of titrated doses of
defined populations of naive, effector, and memory CD8 T cells from the
clone-4 TCR-transgenic mice provided protection against pulmonary
infection with a lethal dose of influenza virus. Naive CD8 T cells were
isolated from the clone-4 TCR-transgenic mice (22)
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 (A/PR/8/34)
(22). Naive CD8 T cells were
CD44low, CD62Lhigh,
CD25negative, and were heterogeneous with regard
to the expression of Ly-6C, as described (21). Effector
cells, primed to produce high amounts of IFN- upon restimulation,
were prepared in vitro as previously described (21).
Effector cells were CD44high,
CD25high, CD62Llow, and
Ly-6Clow highly activated blast-size cells. The
resting CD8 memory cells were generated by adoptive transfer of in
vitro generated Tc1 effectors from the clone-4 TCR-transgenic mice into
adult-thymectomized, irradiated, bone marrow-reconstituted animals, as
described previously (21). Similar numbers of highly
enriched CD8+Vß8+
populations, as defined, were adoptively transferred into hosts
infected intranasally with 10 LD50 influenza
virus (Fig. 1
), and survival was
monitored for 21 days. In all experiments, hosts, which did not receive
passive cell transfer, died between day 7 and day 10 after infection
and cell transfer. When 107 naive CD8 T cells
from the clone-4 TCR-transgenic mice were adoptively transferred into
influenza-infected hosts, recipient animals died between days 6 and 8,
and a slightly accelerated death rather than protection of the adoptive
hosts was observed (Fig. 1a). In contrast, Tc1 effectors
protected very efficiently against influenza virus infection at
107 effector cells transferred (Fig. 1b), and so did 107 resting Tc1 CD8
memory cells (Fig. 1c). The observed protection was cell
dose dependent, because when 106 CD8 T cells were
transferred, only one animal survived when Tc1 effector was
transferred, and all recipients of 106 Tc1 memory
cells died. Thus, the adoptive transfer of naive CD8 T cells was
detrimental for lethally influenza-infected hosts, whereas the same
transferred numbers of effectors or memory cells promoted host
survival.
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To further investigate the relative effectiveness of naive,
effector, and memory cells to protect against pulmonary influenza virus
infection upon adoptive transfer, we determined the pulmonary virus
titer (Tables I and
II). Lungs of recipients of
107 naive CD8 T cells, 107
Tc1 memory cells, or from hosts, which had not received passive cell
transfer, were assayed on day 4 after pulmonary virus infection and
cell transfer (Table I). On day 4, Tc1 memory cells had only slightly
reduced the pulmonary virus titer, whereas no obvious differences were
seen between viral titers in lungs of the adoptive hosts of naive CD8 T
cells and of hosts without passive cell transfer. At this time point,
Tc1 effector cells, however, had already reduced the pulmonary virus
titer (Table II). When kinetics experiments were conducted, the
adoptive transfer of naive CD8 T cells did not decrease, but even
slightly increased the pulmonary viral titer on day 5 as compared with
the mice, which had not received transferred cells (data not shown),
and most recipients of naive cells were already dead on day 7 after
infection (Fig. 1
a). Adoptive transfer of effector cells,
however, resulted in virus clearance at day 5 after virus infection
(data not shown). We also determined the amount of the viral Ag H1N1 by
immunohistochemistry in recipients of Ag-specific naive or memory cells
(data not shown). On day 3 after infection and adoptive transfer, still
similar amounts of positive staining were detected in recipients of
naive or memory cells, and there was still no significant difference
between them on day 5. On day 7, however, there was almost no viral
H1N1 protein detectable in recipients of memory cells, all of which had
survived. In contrast, a high intensity of viral protein was detectable
in the few recipients of naive cells that had survived to day 7. No
H1N1 protein was detectable in sections from noninfected animals or in
isotype control-stained sections. Thus, viral clearance mediated by CD8
memory cells occurs between days 5 and 7, later than by effector cells,
which occurs between days 3 and 5. A high viral load is found in the
recipients of naive cells at all time points (days 3, 5, and 7) until
they die, which occurs at about day 7.
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Since contact-dependent lysis at the site of infection has been
shown to be the main effector mechanism of CD8 T cells during influenza
virus infection (24), homing of high numbers of cytotoxic
effectors to the infected lung epithelium might be crucial for their
protective value. We adoptively transferred 107
naive CD8 T cells, Tc1 effectors, or Tc1 memory cells (donor cells were
Thy-1.2 positive) into influenza-infected Thy-1.1 BALB/c recipients and
determined the percentages of donor cells on days 1, 3, 4, 5, and 7 in
the bronchoalveolar lavage by staining for Thy-1.2 and CD8 expression
and subsequent FACS analysis (Fig. 2).
The day 1 time point was conducted approximately 15 h after virus
infection and cell transfer, and it is likely that at this time point,
most donor cells might have still been in the circulation and have not
yet homed to the harvested organs. The absolute cell numbers of
recovered donor cells from influenza-infected and not infected
recipients were assessed by calculating the percentages from the FACS
profiles and multiplying by the total number of cells per organ (Fig. 3). Effector cells were found in high
numbers in the bronchoalveolar lavage already on day 3 after infection
(350 times more as compared with donor cell numbers of naive cells, and
72 times more as compared with donor cell numbers of memory cells), and
at this time point, recipients of Tc1 effector cells had a lower viral
titer in their lungs than infected animals without cell transfer. Donor
cells derived from Tc1 memory cells, however, started to increase in
numbers in the bronchoalveolar lavage on day 4, and subsequently were
found in high numbers on days 5 and 7 after influenza virus infection.
On day 4, donor cells derived from naive CD8 T cells from the clone-4
TCR-transgenic mice had entered the lung in small numbers, and 7-fold
lower donor cell numbers as compared with memory cells were found on
day 5. In all experiments, several infected animals that had received
naive CD8 T cells had already died by day 7 (Fig. 1), and at this time
point, only the few surviving animals could be analyzed. Interestingly,
when donor cell numbers were determined in the absence of infection,
only cells derived from effector cells were found in the
bronchoalveolar lavage in the absence of other inflammatory cells
(Fig. 3).
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Next, we established, whether naive, Tc1 effector, and Tc1 memory
CD8 T cells had distinct distribution patterns in the draining TBLN and
the spleen. In the experimental protocol that we used, however, we
could not distinguish between proliferation, cell death, and cell
migration, and could only calculate the absolute donor cell numbers per
organ. We analyzed the donor cell numbers in the TBLN (Fig. 4a) and spleen (Fig. 4b) in parallel to the bronchoalveolar lavage shown in Figs. 2 and 3. Donor cells derived from memory or effector cells recovered
from the draining lymph node of influenza-infected recipients increased
over time. Memory cells started to expand by day 3 after infection, and
on day 7 ten times more donor cells were derived from memory cells
compared with naive cells. In the draining lymph nodes, only low cell
numbers of all populations were found in the absence of influenza virus
infection (Fig. 4a). Interestingly, only low numbers of
effector cells were found in the lymph nodes at early time points,
presumably due to the fact that at the time of adoptive transfer,
effectors did not express the lymph node homing receptor CD62L
(21). Effector cells, however, were found in the spleens
(Fig. 4b) in high cell numbers, regardless of whether the
recipients were infected or not, whereas naive cells and memory cells
were found in comparably low cell numbers in the sampled organs in not
infected animals. To investigate the contribution of proliferation of
the adoptively transferred cell populations on the absolute donor cell
recoveries in uninfected mice, we adoptively transferred CFSE
dye-labeled cell populations. CFSE fluorescence intensity is lost by
half upon cell division, and thus we could monitor cell division. We
found that only effector CD8 T cells, but not naive and to a minor
extent memory populations went through multiple divisions after
adoptive transfer (data not shown).
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in vivo
We have reported previously that Tc1 memory cells, but not naive
CD8 T cells, produced high amounts of IFN- after restimulation with
specific Ag in vitro (21). To compare the quality of the
transferred naive or memory CD8 T cells with regard to their potential
to produce IFN- in vivo, we stained for intracellular IFN- when
donor cells derived from naive, effector, and memory cells were
harvested from different organs on day 3 of influenza virus infection
and cell transfer (Fig. 5). After
stimulation with HA peptide-loaded APCs overnight, donor cells derived
from memory cells were not only found in higher numbers, but already
made high amounts of IFN- when harvested from the draining lymph
nodes and spleens. In contrast, donor cells derived from naive CD8 T
cells stained only slightly positive for intracellular IFN- at this
time point. On day 3, only effector cells producing high amounts of
IFN- were found in the bronchoalveolar lavage. The capacity to
produce IFN- by effectors was even more pronounced when the 5-day
time point was analyzed (data not shown). On day 5, donor cells derived
from memory cells still stained more positive for intracellular IFN-
than donor cells derived from naive cells (data not shown) and were
found in higher numbers in the bronchoalveolar lavage (Fig. 2).
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Next, we investigated the change of expression of CD44 (Fig. 6A) and CD62L (Fig. 6B), both of which were suggested to play a role in cell
migration into inflamed tissues (25, 26), on donor cells
derived from naive and memory CD8 T cells over time in
influenza-infected animals. In the spleen, naive CD8 T cells started
out as CD44low cells, as described
(21), and up-regulated expression of CD44 over time. The
expression of CD44 and CD62L on day 1 in the animal was similar to the
expression at t0 before adoptive
transfer (data not shown). Similar kinetics results were obtained when
donor cells harvested from the draining lymph nodes were analyzed (data
not shown). No up-regulation of CD44 was observed when naive CD8 T
cells were parked in hosts in the absence of influenza infection (data
not shown). In contrast, memory cells were
CD44high to start and did not change levels of
CD44 during infection. Both naive and memory donor CD8 T cells started
out as mostly CD62Lhigh cells and down-regulated
CD62L over time in infected animals. In the bronchoalveolar lavage,
however, all donor cells, regardless of whether derived from naive and
memory CD8 T cells, were CD44high and
CD62Llow on day 5 after infection (Fig. 7). Effector CD8 T cells were
CD62Llow on transfer and remained
CD62Llow in the adoptive host (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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upon restimulation early during infection.
In this study, we investigated the performance of three homogenous
Ag-specific CD8 T cell populations during different stages of their
differentiation highly defined with regard to function and phenotype.
Since contact-dependent lysis was shown to be the main effector
mechanism of CD8 effector cells during influenza virus infection
(24), homing of high numbers of cytotoxic T cells to the
influenza virus-infected lung epithelium to get in close contact with
their targets seems crucial. A number of studies (reviewed in Refs.
5, 10, 27), including our own previous studies
(9, 21), have described phenotypic features of naive,
effector, and memory CD8 T cells. The differences in the expression
levels of activation and adhesion surface molecules could make a major
contribution to the function and migration of these cell populations.
In our previous study (21), naive cells from the clone-4
TCR-transgenic mice were found to be CD44low,
CD62Lhigh, and CD25negative
cells. Effectors, primed in vitro for high IFN- production,
up-regulated CD44, down-regulated CD62L, and expressed high levels of
CD25. Resting memory cells generated in this study, however, were
CD44high, CD62Lhigh, and
CD25negative. In addition, LFA-1
(21) and certain carbohydrates (28) were
found to be differentially expressed on naive and memory CD8 T cells.
The surface molecules, which enable a cell population to enter the
lung, are not well defined. In the experimental system we used, we
determined absolute donor cell numbers recovered from different organs,
but could not distinguish between cell proliferation, cell death, and
migration. We found that only effector CD8 T cells entered the lung
with rapid kinetics and appeared there in high cell numbers, whereas
naive CD8 T cells and resting memory CD8 T cells were first seen to
increase in cell numbers in the draining lymph node, and subsequently
entered the lung as CD62Llow, blast cells (Figs. 3 and 7). In other studies, activated memory cells were protective,
while resting memory cells failed to combat infection at a local site
(17). Under the conditions we used, resting memory cells
were able to protect, even though they had to be activated before they
entered the lung. Only CD62Llow cells were found
in the lung lavage (Fig. 7), and we speculate that the expression of
CD62L, which was shown to be the lymph node-homing receptor (29, 30), directed cells to the lymph node, where they stayed until
down-regulation of CD62L had taken place. In some studies, CD8 memory
cells were found to be more heterogeneous with regard to CD62L
expression (20), and it is likely that these
subpopulations might differ with regard to their recirculation
patterns. In contrast, most CD4 memory cells generated after priming
with keyhole limpet hemocyanin from the spleen were found to express
low levels of CD62L (31), and lymph node entry of CD4
cells was shown to depend on CD62L expression (29). Thus,
CD4 and CD8 memory cells might differentially enter the lymph node,
depending on their expression levels of CD62L.
Recently, the requirement for CD44 on activated T cell extravasation into the inflamed peritoneal cavity after injection of SEB was demonstrated (26), and CD44-mediated extravasation was found to depend on its interaction with hyaluronate. No hyaluronate binding was seen with resting CD69negative cells expressing CD44, suggesting that CD44-mediated hyaluronate binding was a feature of activated and not resting cells. In our study, both effectors and memory cells expressed high levels of CD44; however, only effectors, but not memory cells, were activated, CD69-expressing cells. Whether CD44 expression on activated CD8 T cells might also mediate extravasation into the lung during inflammation has to be determined. In addition to adhesion molecules such as selectins and integrins, certain chemokines were shown to attract lymphocytes expressing chemokine receptors, and chemokine gradients might influence directions of lymphocyte migration. CD4 lymphocytes were shown to differentially express patterns of chemokine receptors, depending on their stages of differentiation and on the cytokines they produce (32, 33). When we tested for the mRNA expression of five CCR (1, 2, 3, 4, 5) and two C-X-C chemokine (2, 4) receptors, we found that Tc1 and Tc2 CD8 effector cells expressed a distinct pattern of chemokine receptors (34). In contrast, naive CD8 T cells did not constitutively express any of the chemokine receptors tested (A.C., unpublished observation), and the significance of this finding has to be determined.
Memory cells, but not naive cells, can secrete a wide variety of
cytokines, and do so with rapid kinetics (21, 35). The
patterns of cytokines produced by Th1 and Th2 CD4 effectors
(35) and Tc1 and Tc2 CD8 effectors (21)
remain stable upon memory generation. CD4 memory cells were found to
up-regulate cytokine mRNA within 2 h (our unpublished
observation), and CD8 memory T cells were shown to stain positive for
intracellular IFN- 6 h after influenza infection (7, 8). In the present study, we have only studied effector and
resting memory Tc1 cells, which were primed in vitro to produce high
amounts of IFN-. We show that resting Tc1 memory cells, but not
naive cells, readily produced high amounts of IFN- upon
restimulation in vitro (21) and in vivo (Fig. 5). Upon
stimulation in vivo, naive CD8 T cells developed into IFN--producing
cells, but needed prolonged stimulation and differentiation (Fig. 5).
Effectors, however, which had entered the lung on day 3, already
stained brightly positive for intracellular IFN-. Although IFN-
has been shown to up-regulate MHC I expression on APC and to
facilitate Ag presentation (36), IFN-
-/- mice were found to successfully clear the
pulmonary influenza virus infection (37), indicating a
less important role of IFN- for influenza virus clearance. It was
also suggested, however, that neutralizing IFN- during onset of
viral infection in the F5-RAG-1-/-
TCR-transgenic animals actually ameliorated disease (11).
In our model, the protective value of the Tc1 effector population did
not require the production of high amounts of IFN- because Tc1
effectors protected regardless whether they were generated from the
clone-4 TCR transgenics on the wild-type (Fig. 1) or from the IFN-
-/- background (34). The
significance of the ability of Tc1 memory cells to produce IFN-
early during influenza virus infection (Fig. 5) has yet to be
determined.
Naive, effector, and memory cells were shown to differ profoundly in
their activation requirements. Naive CD4 T cells and CD8 T cells
require additional signals to TCR ligation to become properly activated
(38) (our unpublished observations). CD4 effector T cells
and CD4 memory cells, however, become activated by a TCR-delivered
signal in the absence of costimulation (39, 40). Resting
CD8 memory cells were also shown to respond to lower peptide
concentrations presented on APC as compared with unprimed cells
(41). We find that CD8 effector and memory cells, but not
naive cells, protected against influenza virus infection (Fig. 1). When
expansion over time was monitored, Tc1 memory cells started to increase
in cell numbers slightly before the donor cells derived from naive
cells, and subsequently higher numbers of donor memory cells were
recovered from the draining lymph nodes and from the bronchoalveolar
lavage (Figs. 3 and 4). In this context, it is important to mention
that pulmonary influenza infection is highly localized to the airway
epithelium, and naive and memory cells, which did not enter the lung
early, were likely to wait till APC-presenting viral Ags had reached
the lymph nodes to encounter the Ag. It is thus tempting to speculate
that the more rapid and more pronounced expansion of the memory
population reflects its less stringent activation requirements.
Throughout the study, a lethal dose (10 LD50) of
the highly virulent influenza virus (A/PR8/8/34) was used. We believe
that a lower, sublethal dose of this virus or infections with a less
virulent strain of influenza virus might require lower numbers of
adoptively transferred Ag-specific CD8 T cells for clearance and might
be cleared with faster kinetics. The cellular requirements for viral
clearance at a sublethal virus dose are currently under
investigation.
Resting Ag-specific Tc1 memory cells generated in our model were found to be noncytolytic when isolated straight out of the animal, but could be restimulated for cytolytic activity upon restimulation in vitro (21). Upon stimulation with specific Ag in vitro, however, naive CD8 T cells from the clone-4 TCR-transgenic mice were also found to develop into cytolytic killers with slightly delayed kinetics as compared with memory cells (data not shown). Whether naive and memory cells differed with regard to the kinetics to develop into cytolytic killers in vivo is not known. When memory cells in LCMV-immune animals were studied, a subpopulation of CD8 blast-size memory cells with cytolytic activity against the peptide-loaded RAM-S cell line was demonstrated (19). The protective value of this subpopulation has not been established, but we assume that these activated cells might resemble effector cells rather than resting memory cells.
During pulmonary influenza virus infection, CD8 T cells expressing either type 1 or type 2 cytokines were described (42), and in general the cytokines produced during influenza virus infection cannot be rigidly categorized into a type 1 or a type 2 response (42, 43). When we dissected the protective value of in vitro polarized Tc1 and Tc2 effectors from the clone-4 TCR-transgenic mice, we found that Tc1 protected more efficiently as compared with Tc2 effectors (34). Thus, the cytokines present in vivo might prime the adoptively transferred naive CD8 T cells into not only Tc1, but also Tc2 cells, which differ in their protective value. Adoptively transferred memory cells, however, were already primed and committed to produce the type 1 pattern of cytokines, and we have shown that cytokine production profiles were remarkably stable during different stages of differentiation (21). Thus, it is likely that the beneficial presumably stable Tc1 profile of memory cells might have contributed to their protective value.
All transferred populations, including activated effector cells,
flourished and expanded in cell numbers upon adoptive transfer in vivo
into infected animals (Figs. 3 and 4). This is in contrast to in vitro
results showing that in most cases restimulation of effector cells with
specific Ag resulted in rapid activation-induced cell death
(13) (our unpublished observation). We found that naive
cells stimulated in vitro with HA peptide-loaded APCs expanded rapidly,
whereas effectors underwent rapid activation-induced cell death (data
not shown). Interestingly, when we restimulated the resting memory cell
populations with APCs loaded with a high dose of the HA peptide in
vitro, we found that memory cells responded with rapid kinetics, but
were also highly susceptible to activation-induced cell death (A.C.,
manuscript in preparation). Thus, in vivo during viral infections,
potent rescue mechanisms might operate to block the activation-induced
cell death seen in vitro at least at the population level, and we are
currently attempting to identify factors involved in this process.
We demonstrate that, upon adoptive transfer, CD8 effector cells are most efficient in the protection against a localized pulmonary virus infection. This finding is correlated with rapid accumulation of high effector cell numbers at the infected tissue site. Memory cells require activation before they enter the lung, but are still effective. Naive cells respond too slowly to provide protection. The results presented in this study have considerable relevance for the design of successful immunotherapy protocols using Ag-specific CD8 T cell populations (44).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Adelheid Cerwenka at the current address: DNAX Research Institute, 901 California Avenue, Palo Alto, CA 94304. E-mail address:
3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; HA, hemagglutinin; MDCK, Madine Derby canine kidney; TBLN, tracheal bronchial lymph node; Tc1, Tc2, CTL-producing type 1 or type 2 cytokines; CFSE, 5-(and-6)-carboxy-fluorescein diacetate succinimidyl ester.
Received for publication February 9, 1999. Accepted for publication September 1, 1999.
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