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-/- Mice Develop Functionally Impaired CD8+ T Cell Responses and Fail to Contain Virus Infection of the Central Nervous System
*
Department of Microbiology, University of Tennessee, Knoxville, TN 37996; and
Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, PA 17033
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-deficient mice develop CD8+ T cells at normal
frequencies when infected with HSV or immunized with OVA Ag but show
impaired cytotoxic and cytokine-mediated effector functions resulting
in enhanced susceptibility to HSV-induced encephalitis. Although these
cells display near normal levels of perforin and Fas ligand, they
remain largely at a naive state as judged by high expression of CD62
ligand and failure to up-regulate activation or memory markers. In
particular, these CD8+ T cells revealed inadequate
expression of the IL-12 receptor, thus establishing a link between CTL
differentiation and LT possibly through regulation of IL-12
receptor. Viruses and tumors could evade immunity by targeting the same
pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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that effect immunity
indirectly (2). Resistance to the defensive action of
CD8+ T cells by disarming them in some way
represents a useful immune evasion strategy for virus-infected and
tumor cells (3). Indeed, certain viruses appear to engage
in multiple acts of CD8+ T cell sabotage,
permitting them to survive in the host and to control levels of tissue
damage they induce (4, 5).
With the recent development of sensitive quantitation methods such as
direct detection of epitope peptide-specific cells by tetramer
staining, it became evident that in some circumstances specific
CD8+ T cells are present but one or more of their
effector functions are blunted. Examples exist for viruses such as HIV
(6), influenza (7), lymphocytic
choriomeningitis virus
(LCMV)3
(8), and corona virus (9) as well as in
cancer immunity (10). In the case of
CD8+ T cell responses in the brain to corona
virus infection, cells detectable by tetramer binding may be present
long after demonstrable Ag has disappeared (9). However,
such cells may lack ex vivo cytotoxicity although they retain
Ag-induced IFN--producing capacity. A similar state of affairs was
also observed in CD4-/- mice that were
persistently infected with LCMV (8). In this instance, the
so-called "Sisyphean" T cells were identified by tetramer staining.
Moreover, even though the CD8+ T cells showed
evidence of recent activation in expressing CD69, they were incapable
of either direct cytotoxicity or IFN production (8). In
the cancer field, instances have also been observed where tumor
Ag-specific CD8+ T cells are present yet lack
effector function (10). However, the same persons may
possess CD8+ cells that function normally against
other Ags such as to EBV-infected cells.
We and others have observed that in mice in which lymphotoxin (LT)
or LT
expression was silenced by gene knockout, most lymph nodes
were absent and their splenic architecture was highly disorganized
(11, 12, 13, 14, 15). The animals may exhibit defects in immune
responsiveness and show heightened susceptibility to some infections as
well as to some tumors (16). In such instances, the
disorganized state of the lymphoid system, serving to preclude adequate
immune induction, was the suspected cause of defective immunity
(17). However, as we demonstrate in this report, an
alternative explanation for defective immunity in
LT-/- mice could lie with the impaired
functional efficacy of effector T cells that are induced. To study this
issue, we compared the relative susceptibility of
LT-/- and control C57BL6 (B6) mice to
infection with HSV. Our results show a markedly enhanced susceptibility
of LT-/- mice to develop encephalitis upon
cutaneous or systemic infection with HSV. Because the
CD8+ T cell response is considered crucial for
immunity to HSV in the nervous system (18) we determined
whether CD8+ T cells were normally induced in
LT-/- mice and whether such cells functioned
effectively. Our findings indicate that the CD8+
T cell response to HSV in LT-/- was induced
efficiently, but their effector function was abnormal, resembling the
Sisyphean cells described by others (8). Accordingly,
although the CD8+ T cell responses in
LT-/- and control B6 mice were comparable,
as measured by tetramer binding or by proliferative responses to
peptide stimulation, the LT-/-
CD8+ cells were functionally defective. The
majority of such cells failed to become CTL or to express intracellular
IFN- upon ex vivo stimulation with Ag. Similar functionally
defective CD8+ T cells were also present in
LT-/- mice immunized with OVA. In addition
to showing functional defects, the
LT-/--specific CD8+
cells expressed an unusual phenotype in that many cells retained CD62
ligand (CD62L) and showed reduced expression of several activation
markers in comparison to B6 CD8+ effectors. The
heightened susceptibility of LT-/- mice to
HSV encephalitis was concluded not to result from impaired HSV-specific
CD8+ T cell induction but to be the combined
likely consequence of functionally defective effectors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Development of mice rendered genetically deficient in LT has
been described previously (11). These original homozygous
mutant female mice were bred to C57BL/6 males obtained from The Jackson
Laboratory (Bar Harbor, ME). The resulting heterozygous female
offspring were bred back to their fathers for at least six generations.
Progeny mice were screened for the presence of the LT
gene (wild type) or the presence of the neomycin insert (mutant) by PCR
on prepared genomic DNA from tail tissue. C57BL/6 SCID founder breeding
mice were a gift from Dr. J. Russell Lindsey (University of Alabama,
Birmingham, AL). All mice were housed in microisolator cages and
maintained in a barrier facility at the Walters Life Sciences Building,
University of Tennessee (Knoxville, TN) and handled in accordance with
institutional guidelines.
Antigens
Peptides. HSVgB (aa 498–505) peptide SSIEFARL, chicken OVA (aa 257–264) peptide SIINFEKL, and OVA265–280 peptide (TEWTSSNVMEERKIKV) were synthesized and supplied by Research Genetics (Huntsville, AL).
Proteins. Chicken egg albumin (OVA) grade VI was purchased from Sigma (cat. no. A2512; St. Louis, MO).
Virus. HSV1 Kos strain and HSV1-17 were grown on vero cell monolayers (cat. no. CCL81; American Type Culture Collection, Manassas, VA), titrated, and stored in aliquots at -80°C until used.
Antibodies
Abs for cytokine ELISA were purchased from Becton Dickinson (San
Diego, CA). Reagents used included the IL-2 standard (cat. no. 19211T),
capture Ab (cat. no. 18161D), and detection Ab (cat. no. 18172D); for
IFN- standard (cat. no.19301T), capture Ab (cat. no. 18181D), and
detection Ab (cat. no. 18112D); and for IL-4 standard (cat. no.
19231V), capture Ab (cat. no. 18191D), and detection Ab (cat. no.
18042D). Fluorescent tagged Abs for FACS staining (also purchased from
Becton Dickinson) included FITC- and PE-labeled IgG1 isotype control
(cat. no. 20604A and 20605A), FITC- and PE-labeled IgG2a isotype
control (cat. no. 20047A and 20075A), FITC- and PE-labeled IgG2b (cat.
no. 23244A and 20075A), FITC anti-IFN- (cat. no. 18114A), CD3e
(cat. no. 01084A), CD4 (cat. no. 09425A), CD8a (cat. no. 01045A), CD11b
(cat. no. 01714A), CD11c (cat. no. 09705A), CD16/CD32 (cat. no.
01241A), CD25 (cat. no. 09985B), CD44 (cat. no. 01225A/01224D), CD62L
(cat. no. 01265B), CD69 (cat. no. 01504A/01505B), CD95L (cat. no.
09071A), and CD154 (CD40L) (cat. no. 09025B/09022D).
Abs for perforin detection (mouse anti-perforin, clone KM585 (P1–8); cat. no. MC-030) was purchased from Kamiya Biomedical (Thousand Oaks, CA), and Ab to DEC-205 was purchased from Serotec (cat. no. MCA949; Raleigh, NC).
Immunization
Wild-type and mutant mice were anesthetized with methoxyflurane (Metophane; Pittman-Moore, Mundelein, IL) and received footpad injection of 106 PFU of HSV1 Kos. In the case of OVA immunization, splenocytes were loaded with OVA protein osmotically as described by Moore et al. (19). Splenocytes (2–3 x 106) were injected i.v. into the tail vein of each mouse.
Virus challenge
Naive 5- to 6-wk-old LT-/- mice and
wild-type mice were injected i.m. with various doses of HSV1 Kos
ranging from 5 x 106 to 1 x
108 PFU. They were examined daily starting from
day 3 for signs of HSV infection that included mobility, wasting, limb
paralysis, and encephalitis. Zoster challenge experiment was performed
as described by Manickan et al. (20). Before challenge the
left flank area was depilated by a combination of hair clipping and use
of chemical Nair (Carter-Wallace, New York, NY). The animals were
anesthetized with metofane (methoxyflurane; Pitman-Moore) and a total
of 20 scarifications were made in a 
4-mm2
area. To such scarifications, 10 µl containing
106 PFU of HSV-1 (strain 17) were added and
gently massaged. Animals were inspected daily for the development of
zosteriform ipsilateral lesions, general behavior changes,
encephalitis, and mortality. The severity of the lesions were scored as
follows: 1+ = vesicle formation; 2+ = local erosion and ulceration of
the local lesion; 3+ = mild to moderate ulceration; 4+ = severe
ulceration, hind limb paralysis, and encephalitis; and 5+ = ultimate
death (mice that were moribund and hence euthanized).
HSV-specific lymphoproliferation
Splenocytes from experimental mice were restimulated in vitro with x-ray-irradiated B6 APCs that were infected with UV inactivated HSV (1.5 multiplicity of infection before irradiation) or pulsed with SSIEFARL peptide (5 µg/ml of gB498-505) or uninfected/unpulsed APCs and incubated for 5 days at 37°C. In some samples Con A (5 µg/ml) was used as a polyclonal positive control and incubated for 3 days. Eighteen hours before harvesting, [3H]thymidine was added to the cultures. Proliferative responses were tested in quadruplicated wells, and the results were expressed as mean cpm ± SD.
OVA-specific proliferation
The procedure is exactly as described above except for the stimulators. The APCs for this experiment were obtained from B6 mice and were pulsed with either OVA 257–264 or OVA 265–280 for CD8+- and CD4+-specific stimulation, respectively. IL-2 (50 U/ml) was added to cultures stimulated for CD8+ T cell proliferation. The incubation was for 5 days with the last 18 h in the presence of [3H]thymidine. The cultures stimulated for CD4+ T cell response had anti-CD8 Ab (at a dose ranging from 0.1 to 10 µg/ml) and the CD8-stimulated culture had anti-CD4 (at a dose ranging from 0.1 to 10 µg/ml).
CTL assays
The CTL assay was performed as described earlier (21). In brief, effector cells generated after in vitro expansion (with peptide or HSV) were analyzed for their ability to kill MHC-matched Ag-presenting targets. They were mixed with the target at varying ratios and incubated for 4 h. The targets included syngeneic cells plus Ag, syngeneic cells without Ag, and allogeneic cells plus Ag. The chromium release results were computed as described elsewhere (21).
Cytokine ELISA
The culture supernatants from the bulk proliferation cultures
without addition of any exogenous cytokines were screened for the
presence of IL-2, IFN-, and IL-4. ELISA plates were coated with
capture Abs for the cytokine mentioned earlier and incubated at 4°C
overnight. The plates were washed with PBS-Tween 20 and blocked with
3% nonfat dry milk for 2 h at room temperature. After washing,
serially diluted samples and standards were added to the plate and
incubated for 2–4 h then washed before addition of cytokine-specific
detection Abs for 2 h. The plates were washed and
peroxidase-conjugated streptavidin (cat. no. 016-030-084; Jackson
ImmunoResearch, West Grove, PA) was added. The color was developed by
adding the substrate solution (11 mg of
2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid in 25 ml of 0.1 M
citric acid, 25 ml of 0.1 M sodium phosphate, and 10 µl of hydrogen
peroxide). The concentration was calculated with an automated ELISA
reader (SpectraMAX 340; Molecular Devices, Sunnyvale, CA).
FACS analysis
Cell suspensions containing 1 x 106 cells were incubated with 1 µg each of a FITC- or PE-labeled Ab. The cells were then incubated on ice for 45 min to 1 h. Cells were washed again with FACS buffer (1 x PBS containing 3% FCS and 0.1% sodium azide) and fixed with 4% paraformaldehyde. Cells were analyzed on Becton Dickinson FACScan using CellQuest software.
Intracellular staining
To enumerate the number of IFN--producing cells,
intracellular cytokine staining was performed as previously described
(22). In brief, 106 freshly
explanted splenocytes were cultured in flat-bottom 96-well plates.
Cells were left untreated, stimulated with HSV SSIEFARL-specific
peptide epitopes (0.1 µg/ml), or treated with PMA (10 ng/ml) and
ionomycin (500 ng/ml), and incubated for 6 h at 37°C in 5%
CO2. Brefeldin A was added for the duration of
the culture period to facilitate intracellular cytokine accumulation.
After this period, cell surface staining was performed, followed by
intracellular cytokine staining using a Cytofix/Cytoperm kit
(PharMingen, San Diego, CA) in accordance with the manufacturer’s
recommendations. For intracellular cytokine staining, the Abs used were
anti-IFN- (clone XMG1.2). All Abs were purchased from
PharMingen. For perforin staining we used anti-perforin Ab instead
of IFN- in the procedure described above, and for analysis we used
CellQuest.
Flow cytometry and tetramer staining
MHC class I (H-2b) tetramers to measure SIINFEKL-specific T cells was produced exactly as previously described (23) by Dr. Altman (Vaccine Research Center, Emory University, Atlanta, GA). MHC class I (H-2b) tetramers to measure SSIEFARL-specific T cells were made precisely as described by Mylin et al. (24). Cells (1 x 106) were suspended in FACS buffer and stained for surface markers with a mixture of Abs that included the tetramers. They were incubated for 45 min to 1 h, washed, and analyzed using Becton Dickinson hardware and software.
RT-PCR for IL-12r2 mRNA
Splenocytes obtained from naive and immunized
LT-/- and B6 mice were purified for
CD8+ T cells by using immunoaffinity columns.
Purified (95% pure as evidenced by FACS analysis)
CD8+ T cells (2 x
106) were immediately transferred to Tri- Reagent
(Molecular Biology, Cincinnati, OH) and mixed well to expose cells to
the lytic action of the reagent. The total cellular RNA was isolated
from the Tri-Reagent cellular lysate by adding chloroform, and
centrifugation followed by ethanol/isopropyl alcohol precipitation of
the aqueous RNA solution according to the manufacturer’s instructions.
Total cellular RNA thus obtained was stored as dry pellets or as
aqueous solution in aliquots at -70°C until used.
Total cellular RNA (2 µg) was reverse transcribed using murine Moloney leukemia virus RT (Promega, Madison, WI) and oligo(dt)18 (301 DNA synthesizer; Applied Biosystems, Foster City, CA). The reaction mix (5x murine Moloney leukemia virus RT buffer, 2 mM dNTP, and 40 U RNase inhibitor; Promega) was incubated at an ambient temperature for 15 min for oligo(dt) priming and then incubated at 42°C for 90 min. The RT mix was then heated at 99°C for 5 min and cooled on ice.
PCR assay
Aliquots of cDNA were used in a 25-µl PCR for detection of
actin, IL-12r2 expression. The primer sequences used were for
actin 5' primer 5'
3' GTG GGG CGC CCC AGG CAC CA; 3' primer 5'3'
CTC CTT AAT GTC ACG CAC GAT; the expected size of the product is 
548
bp, for IL-12r2 5' primer 5'3' GGG AGT ACA TAG TGG AAT GGA; 3'
primer 5'3' GCG TCG GTA CTG AAT TTC GCA; the expected product size
352 bp. The reaction mix consisted of 2 mM
MgCl2, 10x buffer A, 10 mM dNTPs-mix, 1 U
Taq (Promega), and 20 pmol primers. The conditions for PCR
amplifications were 94°C (denaturation) for 90 s, annealing at
55°C for 60 s, and extension at 72°C for 120 s. For each
message, the PCR was conducted for 35 cycles. The PCR products were
analyzed on a 2% agarose gel, ethidium bromide stained, and
photographed. The intensity of target band was measured by densitometer
(Bio-Rad, Richmond, CA) and analyzed using Molecular Analyst software.
Ratio of mean intensity of IL-12r2 to actin bands is
reported.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice are more susceptible to HSV infection
Groups of age-matched LT-/- or control
B6 mice were infected with differing doses of HSV-1 and examined daily
for clinical signs correlating to infection of the nervous system. Two
routes of infection were used. Mice were either infected intradermally
in the skin of the flank, an infectious route that results in cutaneous
lesions and encephalitis (20), or, alternatively, mice
were infected via i.m. injection. LT-/- mice
were more susceptible than B6 animals in each case (Fig. 1
, a and b) with
LT-/- mice succumbing to lesion
manifestations and/or encephalitis more quickly and at lower doses of
virus than required to induce comparable effects in B6 mice. Moreover,
whereas B6 mice often manifested clinical signs of infection followed
by recovery this was not observed in LT-/-
mice. With respect to HSV pathogenesis, control of infection within the
nervous system of mice is considered to be orchestrated primarily by
CD8+ T cell function (18).
Consequently, an assessment of CD8+ T cell
activity was further analyzed.
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-/- mice
LT-/- and control B6 mice were
infected with a sublethal dose of HSV-1 on two occasions (days 0 and
14). Animals were sacrificed on day 21, and their splenocytes were
analyzed by several assays for CD8+ T cell
responses to an immunodominant peptide recognized by B6 mice
(SSIEFARL). As is evident in Fig. 2, nylon wool nonadherent T cells from both mouse strains proliferated to
an equal extent when stimulated with SSIEFARL peptide presented on
normal B6 APC. However, when LT-/-
splenocytes were used to present the SSIEFARL peptide, the
responses of both LT-/- and B6
responders were reduced 2-fold when compared with those occurring
when B6 splenocytes were used to present peptide. Such diminished Ag
presentation by LT-/- splenocytes might be
associated with a reduced number of dendritic cells. Thus, the
percentage of DC in B6 mice was shown, using the DEC205 marker, to be
4.2%. However, in LT-/- splenocytes, the DC
percentage was only 0.8%.
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-/- (6.1%) and B6 animals
(5.9%) (Fig. 3). This pattern of results
was also observed in samples analyzed 8 wk after immunization (Fig. 3).
However, in contrast to the results with proliferation and tetramer
staining, marked differences were evident in the effectiveness of HSV
and peptide-specific CTL effector gen-erated by splenocytes from
HSV-immune LT-/- and B6 mice following in
vitro stimulation for 5 days with virus or peptide presented on normal
B6 APC. The experiments show that the B6 CTL response was significantly
higher than were responses generated by
LT-/- splenocytes. This pattern of results
was evident when either HSV or peptide targets were used to measure the
CTL response or, alternatively, if virus or peptide was used during in
vitro restimulation. On average, the defect in peptide-specific CTL
activity in LT-/- cells was 4-fold (Table I).
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, the unresponsiveness
of LT-/- splenocytes to HSV was also evident
when measured by peptide-induced intracellular IFN- expression. This
now well-accepted assay of CD8+ T cell function
in which primed cells are stimulated briefly in vitro with peptide in
the presence of brefeldin A (22), indicated that
LT-/- CD8+ T cells
responded to HSV infection significantly less effectively (10-fold)
than did control B6 CD8+ T cells.
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-/- mice
To extend analysis of the functional defect of
CD8+ T cells in LT-/-
mice, responsiveness to OVA was measured because reagents for this
system are more available to quantify both CD8+
and CD4+ peptide-specific T cell responses. Thus,
LT-/- and B6 mice were immunized with OVA
protein using osmotically loaded normal splenocytes as described by
Moore et al. (19). Immunization on two occasions generates
OVA peptide-specific CD8+ CTL in B6 mice as
originally detected by cytotoxicity of in vitro expanded cells against
EG7 target cells (19). As is evident in Table II, immunization of both B6 and
LT-/- mice induced a cell population that
was specifically cytotoxic to EG7 cells. However, the levels of lysis
and computed LU expressed by the LT-/-
population were markedly less than those observed in the B6 population
(average of three experiments, 11 LU for
LT-/- and 79 LU for B6). Even though the
levels of cytotoxicity for the two populations differed, measurement of
the number of SIINFEKL peptide, the immunodominant
CD8+ epitope in OVA protein recognized by B6 mice
(19), specific CD8+ T cells by
tetramer binding revealed closely similar responses in both
LT-/- and B6 mice (Fig. 5, top). Likewise, the
proliferative responses of nylon wool nonadherent T cells from the two
groups of mice to SIINFEKL peptide and to OVA 265–280 peptide (a
CD4+ recognizable peptide: Ref. 25)
presented on B6 APC were quite similar (Table III). Such data indicate that the
induction of CD8+ and CD4+
T cell responses to OVA was basically essentially normal in
LT-/- mice. However, although
CD8+ T cells proliferated in response to peptide
stimulation, the majority of cells failed to differentiate into CTL
effectors.
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, bottom, SIINFEKL-specific
CD8+ T cells in LT-/-
mice also displayed a marked defect (>20-fold) in function in terms of
intracellular IFN- expression. Thus as detectable by both
intracellular IFN- expression in cells briefly expanded in vitro,
and also by measurement of IFN- levels in the supernatant of
SIINFEKL peptide-stimulated splenocytes (Table III), the
CD8+ produced IFN- response was diminished
8-fold in the LT-/- population.
In an unrelated system, functionally defective
CD8+ T cell responses to certain epitopes
occurred in conditions in which helper T cell responses were absent
(8). In LT-/- mice helper T
cell responses, as measurable by the response to the OVA 265–280
peptide, appeared to be normal as judged by similar levels of IL-2
generated in the supernatant of class II peptide-stimulated splenocytes
from B6 and LT-/- mice. However, the
production of IFN- levels in the supernatant of
LT-/- mice was reduced by 2-fold in
comparison to B6 mice. This minor deficiency appears small when
compared with SIINFEKL-stimulated CD8+ T cells,
where the diminished IFN- response was as great as 8-fold.
To further assess the role of helper cells and their contribution to
the functional defect of the LT-/-
CD8+ T cell response, other experimental
approaches were performed. Initially, comparisons were made between
CTLs generated by SIINFEKL-primed cells to stimulation by EG7 cells in
vitro in the presence or absence of added IL-2. This procedure resulted
in an enhancement of the CTL by primed B6 cells (13%). With
LT-/--primed cells, the response was
similarly increased (10%), but the final CTL responses of
LT-/- cells were still much more reduced
than those mounted by B6 cells without IL-2 supplementation (data not
shown).
LT-/- CD8+ T cells generate CTL in
vitro
If the failure to fully differentiate into
CD8+ T cells by the majority of
LT-/- effectors is a primary defect not
reflective of abnormal splenic architecture and the absence of lymph
nodes, then the defect might also manifest when naive
CD8+ T cells are induced to differentiate into
CTL in vitro. To test this idea, splenic CD8+ T
cells from LT-/- and B6 mice were isolated
by selection on an immunoaffinity column and stimulated in vitro by
procedures shown previously to result in OVA-specific CTL induction
(26, 27). Accordingly, nylon wool-enriched nonadherent
splenocytes were stimulated in vitro under optimal conditions
(determined previously) with OVA protein-loaded B6 APCs. The CTLs thus
generated were tested for activity against EG7 target cells, and the
results of two separate experiments are shown in Fig. 6. These results do show a diminished
responsiveness of LT-/- effectors, which
were 2-fold less cytotoxic than those of B6
CD8+ T cells. This indicates that some, but not
all, LT-/- CD8+ T
cells may have a primary differentiation defect, but the major effect
(accounting for the defect following ex vivo expansion) may result from
failure of appropriate costimulation.
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To determine whether LT-/-
CD8+ T cells expressed a phenotype that
distinguished them from B6 CD8+ T cells, animals
of both strains were immunized with osmotically loaded splenocytes.
Seven days after the second immunization splenocytes were isolated,
stimulated briefly with SIINFEKL peptide, and the subsequent expression
of various surface molecules present on SIINFEKL tetramer plus cells
was measured. The data in Fig. 7 show
some differences in the phenotypic markers expressed by the majority of
LT-/- compared with B6 cells. Accordingly,
many of the LT-/- tet+
CD8+ T cells retained CD62L expression (45%),
whereas this marker was absent on the B6 population. The early
activation marker CD69 was present on 25% of the B6 cells, but only
present on a minority of LT-/- T cells
(7%). Other activation markers analyzed included CD25, CD40L, and
CD44. In all cases LT-/-
tet+ cells had reduced expression of these
markers when compared with B6 control cells.
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-/- mice showed that a majority of cells
express an abnormal activation phenotype. In contrast, when comparing
the cytotoxicity components of LT-/- and B6
tet+ CD8+ T cells,
significant differences between them were not detected. Accordingly
80% of LT-/- cells and 90% of B6 cells
expressed FasL. In addition, the expression of perforin in
tet+ CD8+ T cells from both
populations, measured by intracellular staining, revealed no
differences in staining intensity between the
LT-/- and B6 CD8+ T
cells (Fig. 8) nor in the total
population of CD8+ T cells stimulated with
anti-CD3. Such data suggest that the impaired killing efficacy of
LT-/- CD8+ T cells was
not due to the absence of perforin or FasL expression.
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2 receptor has been shown to be involved
during differentiation of Ag-stimulated T cells into effectors
(28), expression of this receptor was compared in
CD8+ T cells taken from OVA-immunized
LT-/- and B6 mice. For this purpose,
CD8+ T cells were isolated by affinity
chromatography, their RNA was extracted, and the expression of
IL-12r2 mRNA was measured and compared with actin mRNA by
semiquantitative RT-PCR. As is evident in Fig. 9, IL-12r2 expression in naive
CD8+ T cells from B6 mice was barely detectable,
but it was strongly expressed in B6 CD8+ T cells
from OVA-immunized animals (under conditions where 15% of the
CD8+ T cells score positive by SIINFEKL-specific
tetramer binding). In contrast, the LT-/-
population from OVA-immunized mice showed markedly less IL-12r2 mRNA
expression (Fig. 9). Such data indicate an abnormal expression of the
IL-12 receptor on LT-/- Ag-stimulated
CD8+ T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-deficient mice, although
having disorganized lymphoid structure, still develop
CD8+ T cells at normal frequencies when infected
with HSV or immunized with the cross-priming OVA Ag. However, such
CD8+ T cells are functionally defective,
displaying minimal CTL activity and IFN--producing capacity. As a
consequence, LT-/- mice may fail to contain
infection by HSV, allowing the virus to spread to the CNS, resulting in
encephalitis and eventual death. Our data add to the growing number of
observations showing that under certain circumstances
CD8+ T cells may be induced, but one or more of
their functional properties are blunted.
Previously, several groups had shown that an organized lymphoid system
appears necessary to generate effective levels of immunity to several
pathogenic agents (16, 17). Mice in which either LT or
LT expression was silenced by gene knockout lack most or all lymph
nodes and have a highly disorganized splenic architecture
(11, 12, 13, 14, 15). Animals are unable to produce germinal centers,
and certain splenic APCs such as dendritic cells may be significantly
diminished in number (29). This disorganized lymphoid
tissue is assumed to be the reason why LT-/-
and LT-/- mice fail to effectively
clear LCMV infection and have markedly impaired CTL responses to the
virus (17). However, as shown in this report, despite the
presence of disorganized lymphoid tissue,
LT-/- mice can still develop
CD8+ T cell responses. These appeared as
quantitatively intact, as measured by tetramer binding or by
proliferative responses to stimulation with a specific peptide
presented on normal APC. Such data indicate that the disorganized
lymphoid tissue does not preclude in vivo immune induction.
Nevertheless, the CD8+ T cells that were induced
generally failed to differentiate into effectors. In fact, the
functional state closely resembles that described by Zajac et al.
(8) observed in mice reactive with a minor epitope of LCMV
(8). These Sisyphean T cells were induced under conditions
of T helper cell suppression. The CD8+ T cells
were readily detectable by tetramer staining but expressed minimal
IFN- upon epitope-peptide recognition. The effector function
deficiency was assumed to represent a form of anergy resulting from
chronic exposure to Ag and inadequate help (8). A similar
circumstance could occur in HIV infection when helper T cells are
declining. In addition, similar observations were made to explain
epitope dominance (30). Under those circumstances, it is
speculated that mimicry by a self-peptide generated a persistent
exposure to Ag and interfered with CTL differentiation
(30).
In our situation, LT-/- mice were neither
exposed to persisting Ags, especially in the OVA circumstance, nor to
diminished T cell help. Thus, at least as measured by proliferative
responses to a CD4+ T cell peptide and levels of
IL-2 released by such stimulation, the responses of immunized
LT-/- and control B6 cells were comparable.
However, a distinct difference was evident in reference to levels of
IFN- produced by CD4+ T cells following
peptide stimulation.
Our results are consistent with the hypothesis that the LT lymphoid
micro architecture fails to provide the appropriate costimulation
environment for CD8+ T cells after they have
recognized Ag. The fact that the spleens of
LT-/- mice contain fewer dendritic cells,
those APC most adept at providing costimulatory signals (31, 32), supports this notion. Conceivably, the shortage of nearby
costimulator-expressing DC and their production of cytokines involved
in differentiating T cells, such as IL-12, could explain the effector
function shortcoming. Adding to the problem could be the fact observed
in this report that the LT-/-
CD8+ T cells had diminished IL-12r2 receptor
expression, thus minimizing their response to any IL-12 released in
their vicinity. Accordingly, IL-12 is considered as a central factor in
the differentiation of CD8+ T cells into potent
effectors (33). Nevertheless, inadequate costimulation
could not provide the total explanation for the effector function
defect. Thus in comparisons of the primary in vitro CTL activity
induced by cultures of naive CD8+
LT-/- or B6 T cells stimulated under
identical conditions, the B6 CTL were 2-fold more effective than
LT-/- CTL.
Supporting the concept that LT-/-
CD8+ T cells fail to differentiate into normal
effectors was the observation that the majority of cells expressed an
unusual activation phenotype. Thus few cells expressed the activation
markers CD44high and CD25, and unlike the
Sisyphean cells of the Ahmed group (8), almost none
expressed CD69. Of particular interest, many of the
LT-/- CD8+ T cells
detectable by tetramer staining retained expression of CD62L, usually a
marker for naive T cells. This could mean that, apart from being
functional underachievers, LT-/-
CD8+ effectors may show abnormal homing
characteristics and fail to gain access, for example, to extravascular
sites of virus infection such as nervous tissue. The fact that few
LT cells expressed CD44high, the adhesion
molecule considered to be involved in endothelial cell adhesion and
extravascular transport (34), is also in line with homing
difficulties. We are currently attempting to trace and compare the
homing activity of effector CD8+ T cells from
LT-/- and control B6 cells to HSV-induced
inflammatory sites in the CNS such as the trigeminal ganglion.
Although our results demonstrate that LT-/-
CD8+ T cells may exhibit an unusual form of
anergy, explanations as to how the absence of LT leads to such a
phenotype are not at hand. However, it should be noted that the lack of
LT resulted in inadequate up-regulation of IL-12r2 expression on
CD8+ effectors. Furthermore, because dendritic
cells produce IL-12 and these cells were diminished in number in
LT-deficient mice, one may speculate that the IL-12r2-expressing
cells present could receive inadequate stimulation to
differentiate into effectors. Alternatively, the depressed CD40L on the
T cells would support a similar outcome. Regardless of how LT
operates the control of IL-12r2 expression, our observations
establish a link between CD8+ T cell
differentiation and LT. Finally, interference with LT regulation
by viruses and tumors might represent a useful as well as novel immune
evasion strategy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Barry T. Rouse, University of Tennessee, Department of Microbiology, M409, Walters Life Sciences Building, Knoxville, TN 37996-0845.
3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; LT, lymphotoxin; B6, C57BL6; L, ligand.
Received for publication August 15, 2000. Accepted for publication October 11, 2000.
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