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8.1/8.2+ T Cells1
* Department of Molecular Genetics, Biochemistry, and Microbiology, and
Veterans Affairs Hospital and Division of Infectious Diseases, University of Cincinnati College of Medicine, Cincinnati, OH 45267
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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usage by flow cytometry and
RT-PCR. V8.1/8.2+ T cells were preferentially expanded;
other clones bore V4, -6, or -11. When V8.1/8.2+
cells were depleted in mice, V4+ T cell clones were
almost exclusively isolated. Measurement of cytokine production
demonstrated that nine of 16 V8.1/8.2+ clones were Th1,
while only three of 13 non-V8.1/8.2+ clones were Th1. In
mice immunized with rHsp60, depletion of V8.1/8.2+, but
not V6+ plus V7+, T cells completely
abolished the protective efficacy of Hsp60 to lethal and sublethal
challenges. Examination of the TCR revealed that a subset of
V8.1/2+ clones that produced IFN-
and were reactive
to F3 shared a common CDR3 sequence, DGGQG. Transfer of these T cell
clones into TCR 
/-/- or IFN--/-
mice significantly improved survival, while transfer of other
V8.1/8.2+ clones that were F3 reactive but were Th2 or
clones that were not reactive to F3 but were Th1 did not confer
protection. These data indicate that a distinct subset of
V8.1/8.2+ T cells is crucial for the generation of a
protective response to rHsp60. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF-,
and the formation of granulomas that function to surround and contain
the infection (3, 4, 5). In patients without underlying
immune dysfunction, this response is usually successful at limiting the
infection and preventing serious, disseminated disease. While the
initial infection is contained, it does persist in the host in a
dormant state within calcified granulomas and is capable of
causing disease by reactivation if immunological dysfunction develops
(2).
Protection against Hc infection can be achieved in mice by immunizing
with Ag from a cell wall/cell membrane extract of cultured yeasts
(6). Subsequent studies revealed that a particular Ag
within the extract, heat shock protein 60 (Hsp60), is immunogenic and
confers protection against infection (7, 8). Moreover, a
region of Hsp60 between aa 172 and 443, termed fragment 3 (F3), is the
protective domain of this protein (9). Studies have
demonstrated Hsp60 to be a potent inducer of cellular immunity and an
immunodominant Ag from Hc as well as other pathogenic organisms
(10, 11, 12, 13). The protective response to Hsp60 and the
resulting efficacy of vaccination are critically dependent on the
presence of CD4+ T cells. It is believed that
activated T cells produce IFN- which activates macrophages enabling
the cells to kill intracellular yeasts.
While extremely diverse overall, T cells are organized as clonal
populations that contain identical Ag specificity as determined by
limited sequences, including 3 complementarity determining regions
(CDR), of which CDR3, in the -chain of the TCR, is believed to
determine Ag-specificity (14, 15). Interaction with a
defined epitope of an Ag by the TCR is required to activate the T cell
to proliferate and generate cytokines, including IFN-. Previous work
with Hc has indicated that biases in TCR repertoires are generated
during the immune response to Hc. In a primary infection of Hc at the
onset of CMI, V4+ T cells are preferentially
expanded in the lungs of mice (16). On the other hand,
V4+ and 6+ cells are
important in secondary infection (17). The expansion of
V6+ T cells also occurs following immunization
with the protective Ag F3, and they play an integral role in the
protective response to this Ag (18).
In this study we analyzed T cells from recombinant Hsp60
(rHsp60)-immunized mice and demonstrated that T cells expressing
V8.1/8.2 are dominant. Among all T cells, regardless of their V
expression, there was wide variation in the ability to respond to F3,
but Th1-producing clones were almost exclusively
V8.1/8.2+. Depletion of
V8.1/8.2+ T cells in mice abrogated the
protective response to rHsp60 immunization in both lethal and sublethal
models. In addition, transfer of a V8.1/8.2 +
clone, which was reactive to F3 and produced IFN-, to / T
cell-deficient mice, restored protection from infection with Hc. These
data indicate that a clonal subset of T cells that express the
V8.1/8.2 region of the TCR is sufficient to generate a protective
response to rHsp60 of Hc.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male C57BL/6 mice and athymic nude mice were purchased from the
National Cancer Institute (Fredrick, MD). TCR
/-/- and
IFN--/- mice were purchased from The Jackson
Laboratory (Bar Harbor, ME). Athymic nude mice were used to produce
ascites. Animals were housed under barrier conditions and were age- and
sex-matched for all experiments. All animal experiments were performed
in accordance with the Animal Welfare Act guidelines of the National
Institutes of Health.
Hc culture
Hc strain G217B yeast cells were cultured and quantified as
described previously (19). Following anesthesia with
isoflurane, mice were infected intranasally (i.n.) with Hc. Infectious
doses were 2 x 106 (sublethal) or 2 x
107 (lethal) yeasts in 
35 µl of
HBSS.
Recombinant Hsp60 and F3 production
Recombinant Hsp60 and F3 from Hc were generated as described previously (9). Briefly, genes expressing each protein were cloned in to the NdeI and BamHI sites of pET19b. Expression of recombinant protein was achieved by transforming Escherichia coli cells with the plasmid and growing the bacteria in Luria-Bertoni broth until an OD600 of 0.4–0.5 was reached. Isopropylthiogalactose was added to cultures at a final concentration of 1 mM and cells were grown for 3 h. Cells were harvested by centrifugation, and the resultant pellet was resuspended in a buffer containing 5 mM imidazole, 500 mM NaCl, and 20 mM Tris-Cl, pH 7.9. Samples were lysed by freeze-thaw cycles, followed by sonication. Soluble and insoluble fractions were separated by centrifugation at 20,000 x g.
The insoluble pellet was suspended and denatured in a buffer containing 6 M urea, 500 mM NaCl, 5 mM imidizole, and 20 mM Tris-Cl, pH 7.9. Denatured material was recovered in the supernatants following centrifugation at 20,000 x g and was subsequently filtered to remove particulate material. The protein was purified by metal-chelate chromatography using a Ni2+-Sepharose affinity column (His-Bind; Novagen, Madison, WI). The resulting protein was eluted with the same buffer used to suspend the insoluble pellet, but with 1 M imidizole. The eluate was dialyzed against buffer containing decreasing amounts of urea, the recombinant protein was concentrated by ultrafiltration, and the protein concentration was measured. Each protein contained <10 pg of LPS/µg of protein.
Splenocyte preparation
Splenocytes were obtained by teasing apart spleens between the frosted ends of two glass slides. Cells were washed three times with HBSS and resuspended in RPMI (BioWhittaker, Walkersville, MD) supplemented with 10% FBS, 1% L-glutamine, 1% sodium pyruvate, 1% nonessential amino acids, 5 x 10-5 M 2-ME, and 10 µg/ml gentamicin if used for the establishment or expansion of T cell lines and clones, but with only 10% FBS, gentamicin and L-glutamine if used in proliferation assays.
T cell line and clone production
Mice were immunized with 100 µg of rHsp60 s.c. and again 2 wk later. Spleen cells were harvested 2 wk after the final immunization and were stimulated with rHsp60 at 5 µg/ml for 2 wk in supplemented RPMI. Following the initial stimulation, fresh medium and Ag were added at 2-wk intervals with fresh irradiated spleen cells that served as APC. Individual T cell clones were obtained following several passages of the parent line by limiting dilution and were propagated as described above with the addition of 5% supernatant derived from rat splenocytes stimulated with Con A (Sigma-Aldrich, St. Louis, MO) for 48 h (20).
Proliferation assay
To each well of a microtiter plate, 2 x
104 resting T cell clones in 0.1 ml and 5 x
105 irradiated splenocytes in 0.1 ml as APC were
added; 50 µl of either medium or Ag (F3 or rHsp60) was added to each
well (final concentration of Ag in each well, 5 µg/ml). The cells
were incubated for 72 h. Cells were pulsed 18 h before being
harvested with the addition of 1 µCi
[3H]thymidine (sp. act., 6.7 Ci/mmol; New
England Nuclear, Boston, MA). Cells were harvested onto glass-fiber
filter paper, and incorporation of radioactivity was measured by a
liquid scintillation counter. Activity is presented as stimulation
index: the cpm of cells stimulated with Ag divided by the cpm of cells
in medium alone. Reactivity to F3 was defined as a stimulation index of
a 
2.5-fold increase in proliferation of cells compared with cells
that were not stimulated with Ag.
V analysis and TCR sequencing
Expression of the V-chain on the surface of T cell clones was
assessed by flow cytometry. Aliquots of each T cell clone containing
105 cells were incubated with anti-CD16
mAb (Fc block; BD PharMingen, San Diego, CA) and one of the
biotinylated Abs against each of the V families for 15 min at 4°C.
After washing three times, the cells were incubated with
streptavidin-PE for 15 min at 4°C and washed three times. Cells were
then fixed with 1% paraformaldehyde. Fluorescence was measured using
the FACSCalibur flow cytometer. V expression was determined by
examining the number of cells within the PE channel divided by the
total number of cells counted.
Once the V family was identified, RNA was isolated from the T cell
clones using RNAzol (Biotecx, Houston, TX) lysis and chloroform
extraction according to the manufacturer’s protocol. cDNA was
synthesized by annealing RNA with 10 ng of an antisense primer specific
to the C region of the TCR and incubating with avian reverse
transcriptase (Invitrogen, La Jolla, CA). The nucleotide sequence of
each TCR cDNA was obtained using a sense primer specific to the
expressed V family and an antisense primer specific to the constant
region of the -chain as previously described (8). PCR
products were purified and subcloned into pCR2.1-TOPO (Invitrogen).
Several colonies were selected and used to isolate DNA. Multiple DNA
samples from each T cell clone were submitted for sequence analysis by
automated sequencing at University of Cincinnati DNA core facility.
Preparation of mAb
Production of mAb was achieved by injection of hybridoma cells
into athymic nude mice. The hybridoma cell line KJ16 (Rat IgG1
anti-V8.1/8.2) was provided by Dr. P. Marrack (National Jewish
Hospital, Denver, CO), RR4-7 (Rat IgG2b anti-V6) was a gift from
Dr. O. Kanagawa (Washington University, St. Louis, MO), and TR3-10 (rat
IgG2b anti-V7) was provided by Dr. I. Weissman (Stanford
University, Stanford, CA). mAb was purified using a protein G-Sepharose
column (Amersham Pharmacia Biotech, Piscataway, NJ).
In vitro depletion of T cells
Mice were injected i.p. with 100 µg of mAb on days 7 and 3 before and on the day of immunization. Animals were subsequently injected once a week until the end of each experiment. Depletion of >95% of the T cell population was confirmed by flow cytometry.
Analysis of cytokine production
Cytokine production was measured using commercially available
ELISA kits for IFN-, IL-4, GM-CSF, TNF- (Endogen, Cambridge, MA),
IL-13 (R&D Systems, Minneapolis, MN), and IL-10 (BD PharMingen).
Cytokine measurements are presented as the amount of cytokine produced
following stimulation, with correction for the levels of production
from unstimulated cells. For purposes of definition, clones designated
Th1 had a ratio of IFN/IL-4 production that was >1, and clones defined
as Th2 had a ratio <1.
Quantitative organ culture
Mice were sacrificed and examined for fungal burden in lungs and spleens. Organs were harvested and homogenized in 5 ml HBSS. Organ homogenates were plated on blood-heart infusion agar plates at multiple 10-fold dilutions and incubated at 30°C until colony growth could be measured. Colony counts are given as mean log10 CFU per organ ± SE.
Adoptive transfer
T cell clones selected based on cytokine production and F3
reactivity were expanded to generate sufficient cells to inject 2
x 106 T cell clones i.v. into each TCR
/-/- mouse. Mice were infected with
5 x 105 Hc yeasts i.n. 8 h later and
observed for survival.
Statistical analysis
To analyze differences in fungal burden, a one-way ANOVA was performed. Under normalized conditions, the Tukey test was used to allow multiple comparisons among the different groups. Differences in survival were assessed by a log-rank test. Fisher’s exact test was used to compare the proportions of T cell clones.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression of Hsp60 T cell clones
To determine the TCR repertoire following rHsp60 immunization, T
cells were harvested separately from two groups of mice, each immunized
twice with 100 µg of rHsp60. T cells were propagated at 2-wk
intervals, and individual T cell clones were isolated by limiting
dilution. Analysis of V expression of each T cell clone was
determined by flow cytometry. Of the 29 clones isolated, the expression
of V8.1/8.2 was present in 56% of the T cell clones. Smaller
proportions of the isolated T cell clones isolated expressed V4, -6,
-10, or -11 (Fig. 1
).
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expression, and the sequence of the Ag recognition
domain, CDR3 (Table I). Several different
J families were represented, with no clear dominant family or
correlation between V and J expression. Several CDR3 domains were
also identified. In each case, clones sharing common CDR3 sequences
shared the same V and J expression. The J sequence among
clones of the same J family did manifest slight differences at the
nucleotide level. Expression of a particular V or J region was
not indicative of CDR3 sequence. The most conserved CDR3 sequences
among V8.1/8.2+ T cell clones were DGGQG (3 of
16) or GVGTP (5 of 16), while analysis of other V families revealed
no shared CDR3 sequences.
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To examine cytokine production by T cell clones, supernatants from
clones were incubated with or without 5 µg/ml rHsp60, harvested after
48 h, and analyzed by ELISA (Fig. 2). Among the 16 clones expressing
V8.1/8.2, nine were considered Th1, and seven were Th2. All of these
clones produced measurable amounts of IFN-, and all but three
produced IL-4. Among non-V8.1/8.2+ clones,
three of 13 produced no IFN-, while three others produced no IL-4.
Unlike V8.1/8.2+ clones, only three of 13
non-8.1/8.2 clones were Th1, while the remaining 11 were categorized
as Th2.
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. In general,
IFN--producing clones also secreted GM-CSF. Similarly, production of
IL-10 and IL-13 by T cell clones directly correlated with IL-4
production. All clones produced detectable levels of IL-13, and only
one did not produce GM-CSF, while IL-10 was not produced by three of
the T cell clones. None of the T cell clones stimulated with rHsp60
released more TNF- than unstimulated cells.
In addition to cytokine analysis, each T cell clone was examined for F3
reactivity (Table I
). T cell clones were stimulated with F3 or rHsp60
to determine whether a correlation existed between TCR sequence and
reactivity. Among the 16 V8.1/8.2+ clones,
nine were reactive to F3. Only two of 7 V4+
clones reacted to F3, while one of two clones expressing V6 or -10
demonstrated reactivity, and neither of the clones expressing V11
responded. Using Fisher’s exact test, there was not a significant
difference (p > 0.05) in F3 reactivity among T
cell clones expressing different V families. Reactivity to F3
correlated with clones expressing the CDR3 regions GVGTP and DGGQG.
T cell repertoire following V8.1/8.2 depletion
To characterize the T cell repertoire in the absence of
V8.1/8.2+ T cells, rHsp60-immunized mice were
administered with a V8.1/8.2-specific mAb, KJ16. T cells were
isolated from spleens 2 wk after the last immunization and cultured
with rHsp60. Twenty-three clones were isolated, of which 75% (15 of
20) expressed V4, and only 25% (5 of 23) expressed either V6 or
-7 (Table II). Several of the
V4+ clones shared CDR3 homology, with six of
the 15 clones expressing QEGTQ, three expressing DGQLG, and four
expressing DRQGA. F3 reactivity was observed among only clones that
expressed the QEGTQ sequence in the CDR3 region, but not other
conserved CDR3 sequences.
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8.1/8.2+ cells was also altered. The number
of clones that produced IL-10, IL-4, or IL-13 was greatly reduced (Fig. 3). Of the 23 clones, 14 produced no
IL-4, 19 clones produced no IL-10, and 16 of the 23 generated no
detectable IL-13. To ensure that altered levels of cytokine production
were not due to problems of sensitivity using the ELISA, clones 20
(Th1), 12 (Th2), and 7 (Th2), which were isolated from
V8.1/8.2-intact mice, were run in parallel to clones isolated from
depleted mice to directly compare levels of production. The results
demonstrated that clones from V8.1/8.2-intact mice had cytokine
production equivalent to that observed in earlier experiments,
but the amount of Th2 cytokines generated from depleted mice was
consistently lower. IFN- production was detected in 11 of 23 clones,
of which all were classified as Th1. GM-CSF was produced by 10 of 23
clones, of which eight were classified as Th1. Unlike the Th2
cytokines, the amount of GM-CSF or IFN- produced among these clones
was similar to the amount generated by rHsp60 clones from
V8.1/8.2-sufficient mice.
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8.1/8.2 cells in Hc infection
To determine whether V8.1/8.2+ cells were
required for the generation of the protective immune response to
rHsp60, immunized mice were injected with mAb to
V8.1/8.2+ cells and
V6+ and V7+ cells or
were given rat IgG. Two weeks after the second immunization, mice were
challenged with a lethal inoculum of Hc (2 x
107) i.n. and observed for survival. All mice
that had not been immunized with rHsp60 succumbed to infection by 11
days postinfection (dpi; Fig. 4A). Similarly, immunized mice
depleted of V8.1/8.2+ T cells did not survive
beyond 12 dpi. Conversely, all mice immunized with rHsp60 and injected
with rat IgG or mAb to V6 and 7+ T cells
survived. At 40 dpi, lungs and spleens were harvested from the
surviving animals and examined for fungal burden. In both groups the
number of CFU was below the level of detection
(102).
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8.1/8.2+
T cells was associated with increased fungal burden, rHsp60-immunized
mice were administered rat IgG depleted of
V8.1/8.2+ or V6 and
V7+ T cells. Two weeks following the second
immunization with rHsp60, mice were infected with 2 x
106 Hc yeasts i.n. and sacrificed at 7 and 14
dpi. Fungal burden in lungs and spleens of mice depleted of
V8.1/8.2+ T cells was not significantly
different (p > 0.05) from that in unimmunized
controls on days 7 and 14 (Fig. 4B). The lungs and spleens
of mice immunized with rHsp60 and given rat IgG or mAb to V6 and
V7 contained significantly fewer CFU (p <
0.05) than unimmunized controls or those given mAb to V8.1/8.2. No
difference in fungal recovery was detected between rHsp60-immunized
mice and immunized mice that lacked V6 and V7
(p > 0.05) on both days 7 and 14.
Depletion of the entire V8.1/8.2+ repertoire
could have a profound effect on the ability to mount an effective
immune response. To ensure that the increase in fungal burden and loss
of survival was attributable to the specific response to Hsp60 and not
a general defect in the immune system, unimmunized mice were depleted
of V8.1/8.2+ cells. These mice in parallel
with nondepleted controls were infected with a sublethal inoculum, and
the fungal burden was determined at 7 dpi. No significant
(p > 0.05) difference was observed between
depleted and intact animals in either lungs or spleens. The mean
log10 CFU (±SEM) in lungs (6.1 ± 0.21) and
spleens (4.7 ± 0.10) from V8.1/8.2-depleted mice did not
differ (p > 0.05) from that in lungs (6.0
± 0.12) and spleens (4.6 ± 0.15) of infected controls.
Adoptive transfer of V8.1/8.2+ T cells
To determine whether individual subsets of
V8.1/8.2+ T cells were capable of conferring a
protective response, individual clones were injected into TCR
/-/- mice that were infected with 5
x 105 Hc. T cell clones were selected based on
F3 reactivity and cytokine production. In control mice that were not
injected with T cells, all mice succumbed to infection by day 32 (Fig. 5A). Mice that were injected
with clone 17 that produced IFN- but was not reactive to F3 did not
survive beyond 30 days (p > 0.05 compared with
controls). Transfer of clone S cells that were reactive to F3 but
produced IL-4 caused an acceleration of infection, and no mouse
survived beyond 22 days (p < 0.05 compared
with controls). Mice that were injected with clone D, which was
reactive to F3 and produced IFN-, prolonged survival
(p < 0.05) beyond that of control animals,
with mean survival reaching 46 days.
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8.1/8.2+ T cell
clones were injected into IFN--/- mice to
determine whether protection could be restored in animals highly
sensitive to Hc infection (5). Clones S, 17, and D were
transferred. All mice receiving no T cells succumbed to infection with
5 x 105 Hc by day 11 (Fig. 5B).
IFN--/- mice that received clone S or clone
17 were similar to mice that received no cells. Transfer of clone D
dramatically enhanced survival (p < 0.05). All
animals survived beyond 35 dpi, and only 40% succumbed by 40 dpi, at
which time the study was terminated. The lungs and spleens of the
surviving mice were analyzed for the presence of Hc, and in two of the
three mice they contained <102 CFU. The
remaining animal had 106 CFU in lungs and
spleen. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-10, and IL-12 (7, 8, 21). In the results presented
here, rHsp60 from Hc caused the expansion of murine T cells that
predominantly express the V8.1/8.2 region of the TCR. Elimination of
V8.1/8.2+ cells abolished the protective
efficacy following immunization with rHsp60 and led to increased fungal
burden in the lungs and spleens of mice. More important, subsets of
V8.1/2+ T cells that were F3 reactive and
produced IFN-, specifically those expressing the DGGQG CDR3 region,
were able to confer a protective response to T cell- or
IFN--deficient mice. Thus, defined populations of T cells are
involved in the generation of the protective response to rHsp60.
The importance of clonal T cell expansion in response to a pathogen
(22, 23, 24, 25, 26) or as a cause of an autoimmune condition
(27, 28) has been documented in several experimental
models. V8.1/8.2+ and
V10+ T cells are expanded during primary and
memory response to lymphocytic choriomeningitis virus
(29). A specific T cell repertoire also has been
identified following infection with Leishmania major and has
been correlated to a bias in Th2 production in susceptible mice
(30, 31). However, the functional properties of the
expanded repertoire in a protective immune response have not been
rigorously addressed. We have reported that
V4+ T cells are preferentially expanded in the
lungs of mice during a primary infection with Hc and that depletion of
these cells causes an increase in fungal burden during primary
infection (16). In secondary infection,
V4+ and V6+ cells are
important in protection (17). In addition to infection
models with Hc, immunization with the protective fragment F3 drives the
expansion of a specific V repertoire. Following immunization with
F3, only V6+ and V14+
T cells are isolated from infected mice. Depletion of
V6+ cells causes a complete abrogation in the
protective response of F3 immunization, indicating that these cells are
functionally required for the protective response to F3
(18).
Immunization with rHsp60 produced a very different profile in the TCR
repertoire compared with immunization with F3 in the same strain of
mouse. The response to rHsp60 was dominated by
V8.1/2+ T cells, whereas
V6+ cells were the preponderant TCR found
following F3 immunization. The contributions of these two dominant V
families to the protective responses induced by each of their specific
Ag were similar. In both, elimination of the V population led to the
abolition of protection. The absence of V6+
cells was associated with an inability to isolate reactive T cell
clones, indicating that this family was necessary for clonal expansion.
A similar finding was observed in DBA/2 mice immunized with peptide
110–121 from sperm whale myoglobin. Elimination of the dominant
V8.2+ family blunts Ag responsiveness in vitro
(32). Conversely, depletion of
V8.1/8.2+ cells from mice immunized with
rHsp60 did not prevent the emergence of multiple Ag-reactive clones. Of
the clones isolated from V8.1/8.2+-deficient
mice, V4+ cells emerged with the highest
frequency. These clones, however, showed a more limited diversity in
terms of CDR3 sequences and V expression. Despite the ability to
generate rHsp60-reactive clones from
V8.1/8.2+-depleted mice, no other V family
was able to compensate for the loss of these cells in vivo.
Despite the multiple differences among T cell clones isolated, there
were common CDR3 sequences found between F3- and rHsp60-isolated
clones. After F3 immunization, 83% of the CDR3 sequences contained a
GG region, and among F3-reactive clones isolated following immunization
with rHsp60, 28% had a GG sequence in the CDR3 region. However, while
the F3 clones were V6+, Hsp60 clones that
contained a GG region of the CDR3 expressed V8.1/8.2 or -10. The
difference in proportion of GG-containing clones could be an example of
differences in the abundance of epitopes presented following
immunization with the intact protein.
V4+ T cell clones were consistently isolated
from mice immunized with rHsp60 in the presence or the absence of
V8.1/8.2+ cells. V4+
cells are preferentially expanded in the lungs of mice during primary
infection, suggesting that this family of T cells recognizes an
important Ag (8). Despite the importance of
V8.1/8.2+ cells following rHsp60 immunization,
these cells were not significantly expanded during primary infection.
While V4+ clones were frequently isolated in
these experiments, the results suggest that these cells, like
V6+ cells, cannot compensate for the loss of
V8.1/8.2+ cells and are not required for the
protective response to rHsp60. Additional experiments have suggested
that the V4+ populations obtained from spleens
in these experiments differ from those expanded in the lung during
primary infection, and that the lung-derived cells recognize an Ag
other than Hsp60, which may also be crucial in generation of the
primary immune response. This result is in agreement with additional
data that most V4+ T cells isolated from lungs
of infected mice show little or no reactivity to Hsp60 (M.
Scheckelhoff, unpublished observation). Studies are currently underway
to determine the Ag specificity and CDR3 sequences of lung-derived
V4+ T cells.
The protective response to rHsp60 requires the production
of type 1 cytokines, especially IFN- (5, 21). Only T
cells expressing V8.1/8.2+ demonstrated higher
proportions of IFN- production among the clones. Only three of the
13 non-V8.1/8.2+ T cell clones generated were
classified as Th1. In addition, while other V families demonstrated
IFN- production, these collective T cell populations were not able
to compensate for the loss of V8.1/8.2+ cells.
This finding does not exclude the possibility that individual Th1
clones that do not express V8.1/8.2 may be protective. Among
V8.1/8.2+ clones, the production of IFN-
alone was not indicative of protection. Transfer of T cell clones
demonstrated that Th1+ that were also reactive to
F3 prolonged survival, while Th1+ clones that did
not respond to F3 did not extend survival beyond that of infected
control animals. These data suggest that following interaction with Ag,
Th1+ V8.1/8.2+ cells are
activated and probably function to activate phagocytes to stimulate
antifungal activity, but only those that produce Th1 cytokines and are
reactive to F3 are involved in the protective response to rHsp60. F3
reactivity, however, is not alone indicative of protective immunity, as
transfer of an F3-reactive 8.1/8.2+ clone that
produced IL-4 and IL-10 into TCR /-/-
mice caused the animals to succumb to infection at a faster rate than
control mice that received no transferred cells. These results support
the finding that excess production of IL-4 and/or IL-10 can be
deleterious to control of Hc infection (4, 21). However,
transfer of these clones does not accelerate the loss of survival in
IFN--/- mice. This finding is most likely
the result of the Th2-biased environment already present in animals
lacking IFN-. Hence, specific clonotypes of
V8.1/8.2+ T cells are capable of generating a
protective response in a highly organized manner that is controlled
through cytokine production among these and other Hsp60-reactive
clones.
Among V8.1/8.2+ clones, 15 of 16 demonstrated
dual production of IL-10 and IFN-. Recent work has demonstrated that
T cells that produce both these cytokines may play an important
regulatory role in the generation of a protective immune response.
Following infection with L. major or Mycobacterium
tuberculosis, IL-10 has been suggested to play a role in
controlling the Th1 response to prevent immunopathology in host
tissues, but may also prevent sterilization of the tissue and allow
dormancy and persistence (33, 34). Evidence that these
cells were pivotal in the immune response was demonstrated by the lack
of clones that produce both IL-10 and IFN- isolated when
V8.1/8.2+ cells were depleted before
immunization. All but five of the clones derived from depleted mice
produced IFN-, and 13 of 22 were classified as Th1, suggesting that
compensation for Th1 production may be possible. However, only two of
the 22 clones isolated from depleted animals produced both IL-10 and
IFN-. This assertion is supported by the finding that neutralization
of IL-10 interferes with the efficacy of rHsp60 immunization
(21).
The results generated here demonstrate that vaccination with the
protective protein Hsp60 in C57BL/6 mice can be directly correlated to
the presence or the absence of V8.1/8.2+ T
cells expressing a specific CDR3 region (in this case, DGGQG). This
finding has important implications for vaccine development for Hsp60 as
well as other protein Ags that require T cell activity. While V
usage may vary depending on genetic differences among populations, the
CDR3 region that determines epitope specificity will be shared among
these cells, and therefore serve as an indicator for a protective
response. In this example, the V family identified is useful in
terms of identification and categorization, but the efficacy remains
dependent on the CDR3. Correlation of a vaccine and a specific,
protective repertoire found within the population can be used as an
indicator for an effective vaccine and reduce the requirement for
extensive efficacy trials. Conversely, proteins that activate TCR
repertoires that are found only in restricted populations will probably
have limited success. In addition, the experiments presented here
indicate that an epitope-specific subset of T cells can successfully
direct the protective response to a single immunogenic Ag, suggesting
that under favorable circumstances, the development of complex
multivariant vaccines may not be necessary.
In summary, a repertoire of T cells expressing V8.1/8.2 are
generated following rHsp60 immunization and are required for the
efficacy of rHsp60-induced protection. Among these cells, a defined
subset that produces IFN- and is F3 reactive has been shown to
prolong survival in mice and is likely to provide the functional
attributes required for protective immunity.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. George S. Deepe, Jr., Division of Infectious Diseases, University of Cincinnati College of Medicine, Cincinnati, OH 45267. E-mail address: george.deepe{at}uc.edu
3 Abbreviations used in this paper: Hc, Histoplasma capsulatum; CDR3, complementarity-determining region; dpi, days postinfection; F3, fragment 3; i.n., intranasal; rHsp60, recombinant heat shock protein 60.
Received for publication May 31, 2002. Accepted for publication September 12, 2002.
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D. M. Lindell, M. N. Ballinger, R. A. McDonald, G. B. Toews, and G. B. Huffnagle Diversity of the T-Cell Response to Pulmonary Cryptococcus neoformans Infection. Infect. Immun., August 1, 2006; 74(8): 4538 - 4548. [Abstract] [Full Text] [PDF]
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M. Scheckelhoff and G. S. Deepe Jr. A Deficiency in Gamma Interferon or Interleukin-10 Modulates T-Cell-Dependent Responses to Heat Shock Protein 60 from Histoplasma capsulatum Infect. Immun., April 1, 2005; 73(4): 2129 - 2134. [Abstract] [Full Text] [PDF]
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M. G. Hartley, M. Green, G. Choules, D. Rogers, D. G. C. Rees, S. Newstead, A. Sjostedt, and R. W. Titball Protection Afforded by Heat Shock Protein 60 from Francisella tularensis Is Due to Copurified Lipopolysaccharide Infect. Immun., July 1, 2004; 72(7): 4109 - 4113. [Abstract] [Full Text] [PDF]
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