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6+ and V4+ T Cells Exert Cooperative Activity in Clearance of Secondary Infection with Histoplasma capsulatum1
*
Research Division, Veterans Administration Medical Center, Cincinnati, OH 45202;
Division of Infectious Diseases, University of Cincinnati School of Medicine, Cincinnati, OH 45267
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR repertoire of C57BL/6 mice
during primary infection with the pathogen Histoplasma
capsulatum. We observed a consistent oligoclonal expansion of
V4+ T cells during the peak of infection and early
stages of resolution. The V4+ family played a role in
protective immunity against the fungus. Depletion of this subpopulation
of T cells hindered optimal clearance of infection from tissues. In
this report we analyze the flux of the V TCR repertoire in the lungs
of C57BL/6 mice with reinfection histoplasmosis. We observed a
significant increase in V6+ T cells on days 7, 10, and
14, the peak and early resolution phases of infection. This skewing was
preceded by an increased number of memory T cells within
V6+ cells. The VDJ sequences of V6 chains were
oligoclonal during the early stages of the infection, suggesting that
the expansion was driven by a small number of Ags. More than 96% of
the expanded V6+ cells were CD4+. Depletion
of V6+ T cells but not V4+ T cells
induced a modest but significant delay in fungal clearance.
Simultaneous depletion of V4+ and V6+ T
cells induced a more pronounced impairment of host resistance. These
studies illustrate the dynamic interactions between V families in
the response to microbial challenge. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T lymphocytes are heterogeneous and can be subclassified according to
the expression of different surface molecules. We have characterized
the role of different T cell subpopulations in resistance against the
Hc infection (6, 7, 8, 9, 10). Recently, we focused on the most
heterogeneous of the T cell surface molecules, the TCR, by using the
variable element of the -chain as a marker to identify T cell V
families involved in resistance against the fungus (11).
We previously reported that T cells bearing the
V4+ chain are expanded in the lungs of mice
during the peak and early clearance phases of primary infection. This
expansion of V4+ T cells has a functional
correlate: deletion of the V4+ subset of T
cells hinders optimal clearance of the fungus from tissues.
Following resolution of primary infection, mice become resistant to a
secondary challenge with the fungus. The cellular immune response in
reexposure histoplasmosis is qualitatively different from that of
primary infection (9, 12). In this study, we analyzed the
V TCR repertoire in the lungs of C57BL/6 mice that had been
rechallenged with Hc by the intranasal (i.n.) route. We found expansion
of V6+ T cells on days 7, 10, and 14 of
reinfection. Sequence analysis of the complementarity determinant
region (CDR)3 revealed oligoclonality during the acute stages after
inoculation. Depletion of V6+ but not
V4+ T cells produced a modest impairment in
fungal recovery on day 7 of infection. However, simultaneous depletion
of V6+ and V4+ T
cells resulted in a more profound impairment in host resistance. These
results indicate that reexposure histoplasmosis is accompanied by a
bias in the TCR repertoire that is distinct from that of primary
infection and that V6+ and
V4+ cells act cooperatively in host resistance
to reinfection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Five-week-old male C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Athymic nude mice, 6 wk of age, were purchased from the National Cancer Institute (Frederick, MD) and used to produce ascites. Animals were housed under standard conditions at the University of Cincinnati animal facility.
Immunization and infection of mice with Hc yeast
G217B Hc yeast cells were prepared and quantified as previously described (13). Mice were lightly anesthetized with methoxyfluorane (Mallinckrodt Veterinary, Mundelien, IL), and the yeast suspension was delivered i.n.; 1 x 104 Hc yeast cells were given as a primary inoculum. Mice were allowed to recover for 8 wk. Secondary infection was initiated by inoculation of 2.5 x 106 yeast cells i.n.
RNA extraction
Groups of mice (n = 6) at different stages of Hc infection were sacrificed, and their lungs were flushed of circulating cells by injection of 20 ml HBSS into the right ventricle in situ. Lungs were recovered and homogenized in 4 ml of RNAzol (Biotecx Laboratories, Houston, TX). RNA was extracted with chloroform and precipitated following the manufacturer’s protocol. RNA was resuspended in nuclease-free water, and the nucleic acid yield and purity were determined by OD260 and OD260/OD280 ratio. Samples were kept at -70°C until being processed.
Quantitative reverse-transcribed PCR of V families
The methodology used to quantify the relative abundance of each
V transcript in lung RNA has been described (14).
Briefly, 6 µg of total RNA were annealed with 50 ng of an antisense
primer complementary to the constant region of the -chain (C1) of
TCR. First-strand cDNA synthesis was performed with avian
myeloblastosis virus reverse transcriptase (Promega, Madison, WI) and
dNTPs. Aliquots of 1 µl of the reverse transcription reaction were
used as template in 20 parallel PCR. Each tube contained a common
nested antisense primer specific to the constant region of the
-chain (C2) and each of 20 V-specific sense primers
(14), dNTPs, and Taq polymerase (Promega).
Reactions were denatured at 94°C for 45 s, annealed at 59°C
for 45 s, and extended at 72°C for 60 s. The number of
cycles necessary to produce a visible signal without saturation was
determined in preliminary experiments. Between 28 and 32 cycles were
used for most samples. The primer sequences have been published
(11).
The abundance of each V-specific PCR product was determined by
Southern blot: 5 µl of each PCR were electrophoresed in 1% agarose
gels, blotted onto nylon membranes (Roche Biochemicals, Indianapolis,
IN), and hybridized with a digoxigenin-labeled DNA probe specific to
the C region of the TCR. After high-stringency washing, 0.1% SSC
and 65°C, the signal was revealed with alkaline
phosphatase-conjugated anti-digoxigenin Fab (GENIUS system;
Boehringer Mannheim, Indianapolis, IN) and the chemiluminescence
substrate Lumiphos (Boehringer Mannheim). The light production was
measured directly with a ChemiImager 4000 instrument (Alpha Innotech,
San Leandro, CA). The relative density index (Rdi) of each band was
calculated using the AlphaEase software (Alpha Innotech) according to
the formula:
 |
Preparation of mAb
The hybridoma cell lines KT4 (rat anti-mouse V4 chain of
TCR IgG2b) and TR3-10 (anti-mouse V7 chain of TCR IgG2b) were
kindly provided by Dr. Garrison Fathman (Stanford University, Stanford,
CA). The hybridoma cell line RR4-7 (rat anti-mouse V6 chain rat
IgG2b) was kindly provided by Dr. Osami Kanagawa (Washington
University, St. Louis, MO). Ascites was prepared in nude mice. The IgG
fraction was purified using a protein G-agarose column (Pharmacia,
Piscataway, NJ), and the concentration of mAb was determined by ELISA
using rat IgG as standard.
Depletion of V+ T cells in vivo
Groups of mice were depleted of V6+,
V4+, or V7+ T cells
by i.p. injection of 150 µg of mAb, produced from each of the
respective hybridomas, on days -7, -3, and the day of infection.
Subsequently, mice were injected twice a week until the end of each
experiment. Control mice were given an equal amount of rat IgG i.p. The
depleting activity of KT4 mAb and TR3-10 mAb has been reported
(11).
Isolation of lung leukocytes
Lungs were excised after flushing circulating leukocytes by injecting 10–20 ml of HBSS into the right ventricle. Lungs were minced apart in 10 ml of RPMI 1640, and a single-cell suspension was obtained by forcing the lung fragments through needles of progressively smaller gauge, followed by filtration through a 60-µm nylon mesh. Leukocytes were purified by a 600 x g centrifugation through a discontinuous 40%/70% Percoll gradient and enumerated with a hemacytometer.
Flow cytometry analysis
Determination of the CD4+ or
CD8+ phenotypes of V4+
and V6+ T cells was performed as follows. Lung
leukocytes from infected and control mice were recovered. Cells were
resuspended in PBS containing 3% BSA and 0.02% sodium azide. Aliquots
of >104 cells were incubated with saturating
concentrations of biotin-labeled anti-CD3, anti-V6, or
anti-V4 (BD PharMingen, San Diego, CA) for 30 min at 4°C.
After extensive washing, cells were incubated with FITC-CD4 and PE-CD8
(BD PharMingen). Finally, streptavidin-allophycocyanin (APC) was added.
The samples were washed three times, then fixed in 2% paraformaldehyde
until flow cytometry analysis was performed. We gated on the
APC+ cells (either CD3, V4, or V6) and
measured the relative proportion of CD4+ cells in
the FITC channel and CD8+ cells in the PE
channel. The results were calculated by dividing the numbers of
double-positive cells by the total number of leukocytes counted by the
instrument. Isotype-matched controls were run with each sample to
determine the color compensation and the position of the gates.
To determine the percentage of V4+ and
V6+ T cells with memory phenotype, lung
leukocytes from infected immune mice and uninfected controls were
incubated with saturating concentrations of anti-CD45 FITC and
anti-CD44 PE-labeled mAb (BD PharMingen). After washing, cells were
incubated with a second layer of biotin-labeled anti-V6 or
anti-V4 followed with streptavidin-APC. Flow cytometry
enumeration was performed by gating on the V4+
or V6+ populations, using the APC channel, and
counting the cells with the phenotype
CD44highCD45dim (memory).
The results were calculated by then dividing the numbers of T cells
with memory/effector phenotype by the total number of
V4+ or V6+ cells
detected, respectively.
TCR repertoire in mice depleted of V6+ T cells
Groups of immune mice were depleted of
V6+ T cells by injection of 150 µg mAb to
V6 as described above. Animals were infected i.n. with 2.5 x
106 Hc yeast cells and sacrificed after 10 and 14
days of infection. A group of V6+-depleted
animals was sacrificed before infection and used as controls (day 0).
The lungs were recovered, RNA was extracted, and the V TCR
repertoire was determined by RT-PCR as described above.
Analysis of CDR3 sequences
The PCR products amplified with V4- and V6-specific
primers were reamplified using a nested common antisense primer
complementary to the constant region of the -chain (C3). The DNA
was purified in agarose gels and ligated in the Topo PCR2.1 cloning
vector (Invitrogen, La Jolla, CA). At least 10 random colonies were
picked for each mouse and submitted for automated sequencing at the
University of Cincinnati DNA core facility. The DNA sequence analysis
was performed using a general purpose spreadsheet package (Quattro-Pro
8; Corel, La Jolla, CA) along with a set of purpose-designed functions
for recognition and translation of the V, J, and CDR3
sequences.
Quantitative organ cultures
Groups of immune mice (n = 10) were injected
with anti-V6, anti-V4, anti-V6 plus anti-V4, or
anti-V6 plus anti-V7 in different experiments. Control
mice were injected with rat IgG. After 7, 14, and 21 days of
inoculation with Hc, mice were sacrificed, and lungs and spleens were
recovered. The burden of Hc was assessed by quantitative cultures as
described previously (13).
Statistics
Analysis was performed with the SigmaStat software (Jandel Scientific, San Rafael, CA). Student’s t test was used to compare the means between two groups. To determine the statistical differences in fungal burden between depleted and control animals, a one-way ANOVA was performed. The Tukey test was applied to perform multiple comparisons among different treatment groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Groups of mice were inoculated i.n. with 104
Hc yeast cells or HBSS and then challenged with 2.5 x
106 yeast cells 8 wk later. The fungal burden was
determined at 7, 14 and 21 days of infection. Prior exposure to Hc
accelerated the clearance of yeasts at each time point that was assayed
(Fig. 1
).
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6+ family is expanded during secondary
infection with Hc
We examined the dynamic changes in the TCR V repertoire in the
lungs of mice during the course of secondary infection with Hc. The
relative abundance of each V TCR message was determined by
semiquantitative RT- PCR and Southern blots, and expressed as Rdi for
each V family, at different days of infection. The results are
illustrated in Fig. 2. Within each V
family, the Rdi of controls was compared with that of mice infected for
3, 7, 10, 14, and 21 days using a Student’s t test. This
analysis was employed because it is sensitive not only to the magnitude
of the difference between control and infected animals but also to the
consistency of the trend among the individual mice. As five multiple
comparisons were made, we applied a Bonferroni correction, setting a
p value < 0.01 as significant. The only V family
that was significantly increased was V6. The Rdi values
significantly exceeded those of controls on days 7, 10, and 14 of
infection. The remainder of the families did not differ significantly
from animals at day 0 of infection by our statistical criteria.
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subfamilies
We reamplified and cloned the V6 PCR products from lungs of
mice at days 0, 3, 7, 10, 14, and 21 of infection; three to five mice
per group were analyzed. Results are summarized in Table I. At day 0, the CDR3 sequences were
diverse. Mice A and C manifested a single sequence found in two
independent clones, encompassing two of 10 sequences. At day 3, mice D
and E showed an equally diverse pattern in sequences, but the ones from
mouse F were clustered into two groups. Four of six sequences contained
the CDR3 sequence RGL followed by the J2.4 element, and two of six
possessed the IAGA sequence followed by the J1.1 element. At day 7,
the CDR3 sequences from mice G and I displayed oligoclonality; 50% of
the sequences from these mice belonged to a dominant motif
characterized by IPP-J2.3 and IARG-J2.3, respectively. Sequences
from mouse H were heterogeneous, but contained several motifs in common
with other mice. At days 10, 14, and 21, the CDR3 sequences were
polyclonal, with the exception of mouse Q at day 21, in which five of
seven independent sequences were identical.
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1.1 element was observed in 10 of 115 (8.7%) CDR3 sequences,
and the motif IARG-J2.3 was identified in 4 of 115 (3.5%)
sequences. None of these profiles were detected in the controls.
Because V4+ cells were prominent in primary infection
and displayed oligoclonality on days 7–14 postinfection, we analyzed
the CDR3 sequences of this population to determine whether there was a
skewing. Sequences from days 0, 3, 7, 10, 14, and 21 are depicted in
Table II. The CDR3 sequences of mice A
and B on day 0 were diverse; all the sequences were either distinct or
present in no more than two independent clones. The sequences from
mouse C were clustered around two motifs. Four of 10 sequences
contained DPGQ followed by the J1.4 element, and 3 of 10 exhibited
the motif DGGQG associated with the J1.2 element. At day 3, the CDR3
differed in all mice except mouse E in which the sequence EWD linked to
J2.6 was evident in three of 10 clones. Mouse F sequences were
diverse, but some of their VDJ amino acid motifs were found in other
infected mice. CDR3 sequences from mice infected for 7 days revealed
that 5 of 10 sequences from mouse H were identical, as well as four of
seven from mouse I and six of seven from mouse J. Sequences at days 10
and 14 postinfection were highly diverse. Only mouse O contained 6 of
10 sequences that were identical: the sequence NG connected to the
J2.6 element. At day 21, the sequences tended to be diverse, with
the exception of mouse R, in which six of 10 sequences contained the
motif DAR followed by the J1.1 element.
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1.1 element was found in one of the sequences from mouse F
at day 3 of infection and in six of the sequences from mouse R at day
21 of infection. DGGQG followed by the J2.6 element was shared
between mouse C in the control group and mouse J. The sequence GPDSSG
followed by the J1.3 element was found in mice F and I, as well as
the sequence PGNG followed by J2.5.
J usage in the V repertoire
During primary infection we observed that the J2.1 element was
highly prevalent (19–24% of V4 sequences) in mice infected for
7–14 days, but was not found in naive mice. In reexposure
histoplasmosis, the J2.1 element is present in 11% of the sequences
at baseline and does not change significantly during the course of
infection (Table III). In contrast, the
J2.4 element was present in 6% of the sequences from uninfected
mice or mice at day 3 of infection, but on day 7 of infection, 28% of
the V4 chains were combined with the J2.4 element, and 18% at
day 10. At day 14 the J2.4 usage was 8%.
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6+ and V4+ T cells from lungs of mice
with reinfection Hc
We sought to determine the distribution of
CD4+ and CD8+ among
V6+ and V4+ T cells
infiltrating the lungs of infected mice. As a reference, we determined
the relative proportion of CD4+ and
CD8+ cells among all, CD3+,
T cells. At day 0, CD3+ comprised 10.9% of the
leukocytes in the lungs, and 54.9% of them were
CD4+. The proportion of lung leukocytes that were
CD3+ peaked on day 7 to 44.2% and subsequently
declined (Fig. 3A). Analysis
of the V6+ cells (Fig. 3B) showed
that this subpopulation encompassed 0.9% of lung leukocytes before the
onset of infection and became 4.2% of lung leukocytes by day 10, the
peak of their expansion. At this day, 96.7% of
V6+ cells were CD4+.
V4+ cells underwent a more modest expansion
(Fig. 3C). At its peak, day 7, 2.2% of lung leukocytes were
V4+, and 93.6% of them were
CD4+.
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6+ and
V4+ T cells with memory phenotype
We asked whether the expansion of V6+ T
cells observed during secondary infection was preceded by an enrichment
of cells with memory phenotype before the secondary challenge with Hc.
The results are shown in Fig. 4. At
day 0, 8 wk after primary infection but before reexposure, 12.5% of
V6+ cells had
CD44highCD45dim phenotype,
in contrast to 4% in naive animals. Among V4+
T cells, the frequency of memory phenotype in naive animals was 4% as
compared with 6% in mice previously exposed to Hc. After secondary
challenge with Hc, we observed an influx of T cells with memory
phenotype that followed a similar pattern between
V4+ and V6+ families.
At day 3 of infection, the frequency of
CD44highCD45dim phenotype
peaked to 22% for V4+ and 18% for
V6+ and declined in both families
thereafter.
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6+ and/or V4+ T cell
depletion in the clearance of Hc secondary infection
Amplification of the V6+ population does
not necessarily correlate with functional importance. To address this
issue, we selectively depleted this population in vivo by injection of
mAb to V6 and evaluated the course of infection. As a control,
separate groups of mice were given rat IgG. In preliminary experiments,
we determined whether administration of the RR4-7 mAb was effective in
depleting V6+ T cells. We harvested leukocytes
from lungs of treated or control mice (n = 6/group)
after 7 days of infection and analyzed the frequency of
V6+ T cells by flow cytometry.
V6+ constituted 12.6 ± 2.8% of lung
leukocytes in infected nondepleted mice and 0.8 ± 0.2% in
depleted mice. Thus, the mAb treatment produced a 94% reduction in the
frequency of V6+ T cells.
Subsequently, we sacrificed depleted or control animals at 7, 14, and
21 days postinfection. We homogenized lungs and spleens and determined
the burden of Hc CFU in each organ. Depletion of
V6+ cells produced a significant 0.6
log10 increase in fungal burden in the lungs of
infected mice at day 7 of infection (Fig. 5A). Organ cultures at day 21
showed an almost complete clearance of infection in all groups and were
excluded from all further analyses.
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4+ T cells could have an impact on fungal
clearance during secondary infection because this subpopulation was
necessary for optimal clearance during primary infection
(11). Mice depleted of V4+ T
cells cleared secondary infection as efficiently as rat IgG-treated
infected controls (Fig. 5
A).
We then examined whether dual depletion of V6+
and V4+ produced a greater alteration in host
resistance than depletion of V6+ cells alone.
As a control, groups of mice were administered mAb to V6 and V7
concomitantly. CFU from lungs and spleens of mice given both mAb to
V6 and V7 did not differ significantly (p
> 0.05) from those given mAb to V6 alone, although both groups
contained higher CFU than the lungs of nondepleted control animals at
day 7 of infection (Fig. 5B). In contrast, simultaneous
depletion of V6+ and
V4+ T cells unequivocally impaired the ability
to clear Hc from either lungs or spleens at days 7 and 14
postinfection.
Effect of V6+ depletion on the V TCR repertoire
We sought to determine whether the depletion of a discrete V
family would induce a compensatory expansion of other V subset(s).
We chose to analyze the repertoire at days 10 and 14 of infection
because all significant V expansions that we have observed during
primary and secondary infection have occurred during these phases of
peak of infection and early clearance. The repertoire of depleted and
control animals was not significantly different at days 10 and 14 of
infection, except for the almost complete abrogation of the V6
signal (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR repertoire during a secondary challenge with the pathogen. We
concentrated on the lungs for two reasons. First, the lung is the
natural portal of entry of natural Hc infection. Second, lung tissues
are useful for studying the dynamic changes of T cell subpopulations.
Uninfected lungs harbor only a small number of resident T cells. After
an infectious challenge, there is a massive influx of inflammatory
cells, including T lymphocytes, into this organ (10).
When we examined the V TCR repertoire in immune animals infected
with Hc, several differences became apparent. Secondary infection with
Hc produced a bias toward a discrete T cell family,
V6+. The dynamics of this expansion were
analogous to those of the V4+ family during
the primary infection. The V6+ family was not
significantly expanded at day 3 of infection, became dominant at days
7, 10, and 14, and returned toward baseline at day 21 of infection. The
bias of the V repertoire occurred between days 7 and 14 of infection
in a fashion that mirrored the influx of cells of lymphoid phenotype
into the infected lungs.
When we sequenced across the CDR3 of V4+ cells
from primary infection, V6+ from secondary
infection, as well as V4+ cells from secondary
infection, we detected a recurrent pattern. Before inoculation (day 0)
and during the early phase (day 3) of infection, the CDR3 sequences
tended to be diverse. During the peak and early resolution phases (days
7–10) of infection, the sequences converged toward a small number of
dominant motifs. Finally, at the later stages of infection (days 14 and
21), the CDR3 sequences became diverse again. The finding of
oligoclonality is suggestive of an Ag-driven process. The diversity of
sequences at the later stages of infection can be attributed to a
nonspecific influx of inflammatory cells, as has been demonstrated in a
model of autoimmune insulitis (15).
The preferential expansion of a particular V family might be
ascribed to any of three circumstances: 1) the presence of a
superantigen, 2) the accretion of independent TCR clonotypes, each
directed toward a different antigenic determinant but all sharing the
same V-chain of the TCR, or 3) the presence of dominant antigenic
determinants that stimulate the expansion of T cell clonotypes with a
limited number of V-chains and CDR3. Oligoclonality in the CDR3
sequences, along with several instances of CDR3 sequences found
independently in more than one mouse, strongly argue for the third
possibility. Reiner et al. (16) described a strong bias
toward the V4 family in the TCR repertoire of mice infected with
Leishmania major. V4+ T
cells derived from these mice were eventually demonstrated to recognize
the immunodominant Leishmania homologue of receptors for
activated C kinase Ag from L. major (17). Our
results suggest that a dominant Ag or Ags from Hc are the driving
stimulus for the bias and oligoclonality in the V repertoire in this
model of infection. Work is in progress to identify the antigenic
targets of V4+ T cells recovered from the
lungs of mice with primary infection and of
V6+ T cells from mice with reinfection
histoplasmosis.
None of the V families that were significantly expanded during
primary infection displayed an increase upon reexposure to Hc. V6,
the only family that exhibited significant amplification during
secondary infection, was not significantly expanded in the lungs of
naive mice infected with Hc. One explanation for these findings is that
the Ag(s) recognized by V6+ cells were cryptic
in primary infection. Conversely, it is possible that both primary and
secondary responses were directed against the same dominant Hc Ag(s),
but T cells specific for this Ag use the V4 chain preferentially in
primary infection and the V6 element in secondary histoplasmosis.
The report by Busch et al. (18) illustrates these
possibilities. In a murine model of listerosis, the TCR repertoire was
biased to the same V families in both primary and secondary
infection in all but one mouse. The lone exception displayed a shift in
the dominant family from V8+ during primary
infection to V2+ during recall immune
response.
A more subtle finding emerged when we retrospectively compared the TCR
repertoire of naive and immune mice before the infectious challenge.
The relative abundance of most V subpopulations is not significantly
different between the groups. Also, the repertoire at day 21,
corresponding to the late clearance phase of the infection, is not
significantly different from the repertoire at day 0. This comparison
has a caveat. The data came from two independent, nonsimultaneous
experiments instead of two groups run in parallel. However, this result
does suggest that the process of primary infection with Hc did not
produce a measurable, permanent alteration of the baseline repertoire.
Only during active infection, days 7–14, were we able to detect
significant deviations within the V repertoire.
Additional phenotypic analysis of V6+ and
V4+ cells revealed several interesting
features. A large majority of each V family coexpressed the CD4
receptor. This finding was in line with the observation that among
CD3+ cells from the lungs,
CD4+ cells comprised the highest proportion. In
addition, we observed that the expansion of
V6+ cells was preceded by an increased
proportion of the memory cells within this subset. In contrast, the
expansion of V4+ T cells during primary
infection did not result in a residual enrichment of memory
V4+ T cells in the lungs before reexposure to
Hc. Nevertheless, after the onset of secondary infection, the flux of
memory cells was similar in both V6+ and
V4+ cells.
Depletion of CD4+ but not
CD8+ T cells impairs fungal clearance in
reexposure histoplasmosis, in contrast with a substantial increase in
mortality during primary infection (9). Simultaneous
elimination of CD4+ and
CD8+ T cells completely abrogates protective
immunity in secondary infection with Hc. In our studies, we asked
whether V6+ T cells were necessary for optimal
clearance of Hc infection in immune mice. Deletion of this family
produced a modest but significant effect on Hc clearance detectable
only on day 7 of infection, whereas animals depleted of
V4+ T cells eliminated Hc as efficiently as
infected controls. Despite the loss of the amplified
V6+ population, the impact on host resistance
was not striking, suggesting that host defenses developed compensatory
mechanisms to combat infection. Because V4+
cells exhibited a prominent influence on the course of primary
infection, we tested the possibility that simultaneous elimination of
V4+ and V6+ cells
caused a more dramatic effect on host resistance. Indeed, the absence
of both families resulted in an unequivocal and pronounced impairment
of host defenses against Hc that was greater than that observed with
elimination of either family. This effect was specific for the loss of
both V6+ and V4+
cells because elimination of the former and
V7+ cells did not impair host defenses more
than depletion of V6+ cells alone. Thus,
V6+ and V4+ manifest
a cooperative interaction in resistance to Hc.
In conclusion, immunization with viable Hc modifies the lung V TCR
repertoire. During days 7, 10, and 14 of infection,
V6+ cells are significantly expanded in the
lungs of immune C57BL/6 mice, which is in contrast to the finding that
V4+ T cells are amplified in primary
infection. Immune mice display an increased frequency of
V6+ T cells with memory phenotype before the
onset of secondary infection. The CDR3 sequences of V4 and V6
chains converge toward oligoclonality during days 7–10 of secondary
infection. Selective deletion of V6+ cells
hinders optimal clearance of the fungus in secondary histoplasmosis.
Moreover, depletion of both V6+ and
V4+ T cells, which represent the predominant T
cell families expanded during secondary and primary Hc infection,
respectively, induced a more profound and sustained defect in the
course of fungal clearance.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. George S. Deepe, Jr., Infectious Diseases Division, University of Cincinnati College of Medicine, 231 Bethesda Avenue, ML 560, Cincinnati, OH 45267-0560.
3 Abbreviations used in this paper: Hc, Histoplasma capsulatum; i.n., intranasal; Rdi, relative density index; CDR, complementarity determinant region; APC, allophycocyanin.
Received for publication April 13, 2000. Accepted for publication November 27, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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a mice: cellular immune responses and susceptibility patterns. J. Med. Vet. Mycol. 31:181.[Medline]
4+ T cells promote clearance of infection in murine pulmonary histoplasmosis. J. Clin. Invest. 102:984.[Medline]
exacerbates primary and secondary pulmonary histoplasmosis by differential mechanisms. J. Immunol. 160:6072.This article has been cited by other articles:
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  A. I. Medeiros, A. Sa-Nunes, W. M. Turato, A. Secatto, F. G. Frantz, C. A. Sorgi, C. H. Serezani, G. S. Deepe Jr., and L. H. Faccioli Leukotrienes Are Potent Adjuvant during Fungal Infection: Effects on Memory T Cells J. Immunol., December 15, 2008; 181(12): 8544 - 8551. [Abstract] [Full Text] [PDF]
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 M. Wuthrich, H. I. Filutowicz, H. L. Allen, G. S. Deepe, and B. S. Klein V{beta}1+ J{beta}1.1+/V{alpha}2+ J{alpha}49+ CD4+ T Cells Mediate Resistance against Infection with Blastomyces dermatitidis Infect. Immun., January 1, 2007; 75(1): 193 - 200. [Abstract] [Full Text] [PDF]
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E. Heninger, L. H. Hogan, J. Karman, S. Macvilay, B. Hill, J. P. Woods, and M. Sandor Characterization of the Histoplasma capsulatum-Induced Granuloma. J. Immunol., September 1, 2006; 177(5): 3303 - 3313. [Abstract] [Full Text] [PDF]
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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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 J.-S. Lin and B. A. Wu-Hsieh Functional T cells in primary immune response to histoplasmosis Int. Immunol., November 1, 2004; 16(11): 1663 - 1673. [Abstract] [Full Text] [PDF]
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 T. R. Johnson, S. M. Varga, T. J. Braciale, and B. S. Graham V{beta}14+ T Cells Mediate the Vaccine-Enhanced Disease Induced by Immunization with Respiratory Syncytial Virus (RSV) G Glycoprotein but Not with Formalin-Inactivated RSV J. Virol., August 15, 2004; 78(16): 8753 - 8760. [Abstract] [Full Text] [PDF]
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M. Scheckelhoff and G. S. Deepe Jr. The Protective Immune Response to Heat Shock Protein 60 of Histoplasma capsulatum Is Mediated by a Subset of V{beta}8.1/8.2+ T Cells J. Immunol., November 15, 2002; 169(10): 5818 - 5826. [Abstract] [Full Text] [PDF]
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G. S. Deepe Jr., R. S. Gibbons, and S. R. Ward Discordance between T-Cell Receptor Expression and Effector Function in Mice Infected with Histoplasma capsulatum Infect. Immun., March 1, 2002; 70(3): 1648 - 1652. [Abstract] [Full Text] [PDF]
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G. S. Deepe Jr. and R. Gibbons V{beta}6+ T Cells Are Obligatory for Vaccine-Induced Immunity to Histoplasma capsulatum J. Immunol., August 15, 2001; 167(4): 2219 - 2226. [Abstract] [Full Text] [PDF]
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