Functional T cells in primary immune response to histoplasmosis
Jr-Shiuan Lin and
Betty A. Wu-Hsieh
Graduate Institute of Immunology, National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China
Correspondence to: B. A. Wu-Hsieh; E-mail: wuhsiehb{at}ha.mc.ntu.edu.tw
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Abstract
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Functional T cells are critical to host defense against infection. It has been reported that functional T cells as determined by their cytokine production represent antigen-specific T cells in infectious disease models. In this study, we enumerated Histoplasma-specific interferon
-producing cells in bulk splenocyte culture and showed that infection with Histoplasma capsulatum, an intracellular pathogen of the macrophage, activated both CD4 and CD8 T cells. The magnitude of CD8 T cell response was lower than CD4 T cell, but the expansion and contraction of both cell types followed the same kinetics. Over 90% of interferon
-producing CD4 T cells and >85% of CD8 T cells expressed CD44hi phenotype. The strong correlation between interferon
production and CD44hi expression was observed not only at the peak of response but also throughout the course of infection. Moreover, a broad spectrum of Vß populations responded to systemic as well as pulmonary infections, suggesting no obvious T cell receptor bias in primary immune response to histoplasmosis. While each Vß population contributed to interferon
production, several specific Vß populations made up higher percentages of interferon
-producing cells. Our study laid the groundwork for further investigations in immune response to histoplasmosis.
Keywords: CD44, histoplasmosis, IFN
-producing cells, specific T cell response, Vß repertoire
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Introduction
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Histoplasma capsulatum is a dimorphic fungus. Microconidia of the fungus are inhaled, convert to yeasts in the host and reside in the phagolysosome of the macrophages. Control and clearance of this pathogen are dependent on intact cellular immune response. Ability of the host to produce IFN
is critical to host defense against the infection (15). In vitro depletion studies have identified CD4 T cells as IFN
producers (5). However, the magnitude and kinetics of CD4 and CD8 T cell responses have not been clarified.
Recent developments have made the enumeration of antigen-specific T cells possible. The most direct assessment includes staining of T cells with tetrameric MHC molecules loaded with specific peptide, intracytoplasmic cytokine staining and ELISPOT assays (613). These methods are sensitive, specific and physiologically relevant (8,9). The numbers of virus-specific CD8 T cells quantified by flow cytometric technique and ELISPOT assay involving short-term peptide stimulation and staining for IFN
are almost identical to those determined by tetramer staining (8,12). Thus, functional T cells as determined by their cytokine production represent antigen-specific T cells in infectious disease models. In this study, we evaluated the Histoplasma-specific T cells in splenocyte bulk culture stimulated with heat-killed yeast cells and employed intracytoplasmic IFN
staining and flow cytometric technique to, first of all, identify the kinetics and the pool size of functional CD4 and CD8 T cells in primary immune response to histoplasmosis. In addition, we studied the phenotypic characteristics of IFN
-producing T cells in the course of infection.
Investigators have used RTPCR methods to identify and quantify TCR Vß usage in animals infected by a pathogen. Results of such studies demonstrated that one or a few specific Vß are preferentially used in response to a specific infection (1420). It has been shown that Vß4+ T cells were preferentially activated in mice intranasally infected with Histoplasma (18,19). In this study, we used a panel of anti-Vß antibodies to analyze Vß usage by functional T cells in pulmonary as well as systemic infections. In contrast to the published data, we found that there was expansion of a broad spectrum of Vß populations of both CD4 and CD8 T cell subsets (18,19). In addition, most expanded populations contributed to IFN
production, though some had a greater contribution than others.
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Methods
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Animals
C57BL/6 breeders were obtained from the Jackson Laboratory (Bar Harbor, ME) and bred at Laboratory Animal Center, National Taiwan University College of Medicine. To ensure genetic stability, new breeders obtained from the Jackson Laboratory are reintroduced every 23 years. Mice at 810 weeks of age were used for all the experiments. Mice were housed in sterilized cages with sterilized bedding and filter cage tops. Mice were fed with sterilized food and water.
Fungus and infection
Histoplasma capsulatum strain 505 yeast phase was cultured at 37°C on brainheart infusion (BHI) agar supplemented with cysteine (1 mg/ml) and glucose (20 mg/ml). Fresh yeast cell suspensions were prepared in RPMI 1640 medium (GIBCO-BRL, Grand Island, NY) for injection. Mice were injected either intravenously (2.5 x 104) or intratracheally (2 x 105) with Histoplasma yeast cells. The size of inocula for both intravenous and intratracheal inoculations was <1/100 of lethal dose (21,22).
To deliver Histoplasma to the lungs, the mice were anesthetized with acepriomazine maleate (0.2 mg/mouse) (Fermenta Animal Health Co., Kansas City, MO). The trachea was carefully exposed using a midline neck incision. A 40 µl inoculum containing 2 x 105 yeast cells was injected directly into the trachea by a sterile 29-gauge needle, followed by injection of 20 µl of air to clear the central airways. The skin incision was closed with surgical staples. Mice were kept warm until recovery from anesthetics.
Reagents
RPMI 1640 medium (GIBCO-BRL) was supplemented with 10% heat-inactivated fetal calf serum (FCS, Biological Industries, Israel), 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM nonessential amino acid, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 x 105 M 2-mercaptoethanol and 25 mM HEPES buffer. All supplements were obtained from GIBCO-BRL. Dulbecco's phosphate-buffered saline (dPBS, Biological Industries) supplemented with 1% heat-inactivated FCS and 0.1% NaN3 (Sigma-Aldrich, St Louis, MO) was used as staining buffer. Dulbecco's PBS supplemented with 1% heat-inactivated FCS, 0.1% NaN3 and 0.1% saponin (Sigma-Aldrich) was used as Perm/Wash buffer.
Preparation of single cell suspensions from lung tissues and mediastinal lymph nodes
The lungs of infected mice were removed at day 14 after infection, teased apart with forceps and ground with two ground glass slides. The solutions were passed through cottoned Pasture pipette to obtain single cell suspension. The mononuclear cell fraction was isolated by separation on discontinuous 4070% Percoll gradients (Amersham Biosciences, Uppsala, Sweden) by a 600 g centrifugation. Cells from six mice were pooled and enumerated by a hemocytometer. The mediastinal lymph nodes (MLN), which drain the lungs, were ground to obtain single cell suspension.
Cell surface and intracytoplasmic cytokine staining
Single cell suspension was prepared from freshly harvested spleen at different time points after infection. One million cells were cultured in flat-bottomed 96-well plates in supplemented RPMI medium or in medium containing 2.5 x 104 heat-killed Histoplasma yeast cells. After 18 h of incubation, 2 µM of monensin (Sigma-Aldrich) was added to the culture and cells were harvested 6 h later. Cells were washed twice with staining buffer and stained with allophycocyanin-conjugated anti-CD4 (clone GK1.5) or anti-CD8 (clone 53-6.7), and FITC-conjugated anti-CD25 (clone 7D4), anti-CD44 (clone IM-7) anti-CD43 activation-associated glycoform (clone 1B11), or anti-Vß antibodies for 30 min at 4°C in the dark. Cells were washed twice with staining buffer, then fixed by Cytofix (4% paraformaldehyde in dPBS) for 20 min on ice and permeabilized by two washes with Perm/Wash solution. PE-conjugated anti-IFN
antibody (clone XMG1.2) in the Perm/Wash buffer was added and cells were left for 30 min on ice in the dark. Cells were again washed twice with Perm/Wash buffer and fixed in staining buffer/2% paraformaldehyde. FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA) was used for cell acquisition. Data were analyzed by CellQuest (BD Biosciences). All antibodies were purchased from eBioscience (San Diego, CA) except 1B11 (BD PharMingen, San Diego, CA) and were used at 0.1 µg per 106 cells. Dead cells were excluded by forward and side scattering.
Quantitation of fungal load in the spleen and lungs
Spleens and lungs were harvested from mice at different time points after infection. The tissues were homogenized in a tissue grinder with 1 ml of RPMI 1640 (Gibco-BRL). One:ten serial dilutions were made and 0.1 ml was plated onto glucosepeptone agar. Mycelial colonies of Histoplasma were counted after incubation at 30°C for 1014 days.
ELISA
Five million freshly isolated splenocytes were cultured in a 48-well tissue culture plate with or without the addition of 1.25 x 105 heat-killed Histoplasma yeast cells. The supernatant was collected after 24 h of incubation and the concentration of IFN
was determined by ELISA (eBioscience).
Staining for TCR Vß repertoire
Freshly isolated splenocytes, lung cells and lymph node cells were prepared as single cell suspensions. One million cells were stained with PE-conjugated anti-CD4 or anti-CD8 and a panel of FITC-conjugated Vß screening antibodies (BD PharMingen). After 30 min incubation at 4°C in the dark, cells were washed twice and fixed until acquired by flow cytometer as described above.
Statistics
Student's t-test was used to compare the cell numbers between infected and uninfected mice.
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Results
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The magnitude and kinetics of specific T cell response
We and others have previously shown that CD4 T cells are major IFN
producers in response to Histoplasma infection (1,5,23,24). It has been shown recently that repeated subcutaneous injections of live Histoplasma in CD4-depleted mice confer resistance to lethal challenge (22), indicating that CD8 T cells alone are protective. However, the kinetics and the pool size of these functional CD4 and CD8 T cells have never been studied. By using intracytoplasmic cytokine staining method, we show that specific CD8 and CD4 T cells produced IFN
in response to Histoplasma infection at as early as day 7 after infection. The peak of response was at day 14 (Fig. 1A and B), and 8.4% and 3.9% of total CD4 and CD8 T cells, respectively, were actively engaged in IFN
production, which means there were 10.1 ± 1.0 x 105 CD4 and 3.0 ± 0.7 x 105 CD8 T cells that were functionally active at this time (Table 1 and Fig. 2). From the data presented here, we estimated that maximally 1/12 of total splenic CD4 T cells and 1/25 of CD8 T cells were specifically activated in response to histoplasmosis. Interestingly, although the magnitude of CD4 T cell response was greater than the CD8 T cells, the kinetics of the expansion and contraction of both cell types followed the same pattern (Fig. 2). The peak of IFN
response as determined by ELISA coincided with that detected by intracytoplasmic staining (Fig. 1C). Moreover, the onset of fungal clearance in lungs and spleens preceded the maximal T cell response and the initiation of the contraction phase of specific T cells occurred before complete fungal clearance (Fig. 2). The contraction phase roughly correlated with the reduction of fungal clearance. At day 35 after infection, where no more fungus was detected, there was still a small number of T cells which remained functionally active.

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Fig. 2. The dynamic relationship between the number of functional T cells and fungal clearance. The fungal counts in spleens (open bar) and lungs (hatched gray bar) of infected mice are the mean values of 610 mice. The mean numbers of CD4+- (open squares) and CD8+- (closed squares) IFN -producing cells are presented in Table 1.
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Most IFN
-producing cells express CD44hi phenotype
To characterize IFN
-producing cells, total splenocytes were stimulated for 24 h and triple stained with anti-IFN
, anti-CD4 or -CD8, and anti-CD25, -CD44 or -1B11 antibodies. CD25 and CD44 are well accepted as T cell activation markers. 1B11 antibody specifically recognizes the activation-associated glycoform of CD43. It has been reported that 1B11 up-regulation in LCMV-specific CD8+ effector T cells strongly correlates with the expression of CTL activity (25). 1B11 expression also distinguishes LCMV-specific CD8 effector T cells from memory T cells (19). At the peak of T cell response to Histoplasma infection, 94% of CD4+ and 85% of CD8+ IFN
-producing T cells expressed CD44hi phenotype and only a fraction (4060%) of them expressed CD25 or 1B11 (Fig. 3A). The strong correlation between IFN
production and CD44hi expression was seen not only at the peak of IFN
response, it was also observed throughout the course of infection, disregarding the total number of IFN
-producing cells at different time points (Figs 1A and 3B). In contrast, the expression of CD25 and 1B11 on IFN
-producing cells was down-regulated at later time points, further illustrating the dissociation of these markers from effector function. Thus, there was a strong correlation between effector cell function and CD44hi phenotype.
Expansion of a broad spectrum of TCR Vß repertoire and IFN
-producing populations in mice systemically infected with Histoplasma
To analyze CD4 and CD8 T cell Vß repertoire, freshly harvested spleen cells from infected mice were stained with a panel of anti-Vß antibodies. The results show that infection did not change the percentage of any of the Vß populations (Fig. 4A). However, there was a significant increase (P < 0.05) of the numbers of CD4 and CD8 T cells in most Vß populations (Fig. 4B). These populations included Vß4, 5.1&5.2, 6, 8.1&8.2, 8.3, 10b, 11, 12, 13 and 17a of CD4 T cells and Vß3, 4, 5.1 &5.2, 6, 7, 8.1&8.2, 8.3, 9, 11, 12, 13, 14 and 17a of CD8 T cells. Thus, our results demonstrate that instead of expansion of one specific Vß population, a broad spectrum of CD4 and CD8 T cell Vß populations undergo expansion in primary immune response to histoplasmosis.

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Fig. 4. Expansion of a broad spectrum of Vß populations of CD4 and CD8 T cells in Histoplasma infection. Splenocytes were collected from uninfected (open squares, n = 3) and infected (closed squares, n = 5) mice at day 14 after infection. Cells were stained with FITC-conjugated anti-Vß and PE-conjugated anti-CD4 or anti-CD8 antibodies. (A) The percentages of each Vß population in CD4 or CD8 T cell subsets before and after infection are compared. (B) The total number of CD4+ and CD8+ T cells expressing Vß in the spleen. The cell number expressing Vß was calculated from the total number of splenocytes, the percentage of CD4+ or CD8+ T cells and the percentage of each subset expressing the specific Vß. Error bars indicate standard deviation of the mean. (*P < 0.05, **P 0.01).
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We went on to determine whether any Vß population was functionally more dominant than others. We determined the percentage of each Vß population contributing to IFN
production. Figure 5(A) and (B) shows that while each Vß population analyzed contributed to IFN
production, several specific Vß populations made up higher percentages of IFN
-producing cells. Vß3, 6 and 8.1&8.2 of CD4 T cells constituted 20.0 ± 3.7%, 12.0 ± 3.8% and 19.3 ± 5.1%, respectively, of CD4+ IFN
-producing cells and Vß3, 5.1&5.2 and 8.1&8.2 of 19.7 ± 5.1%, 23.6 ± 8.0% and 22.6 ± 9.4% of CD8+ IFN
-producing cells (Table 2).
Comparing IFN
-producing Vß populations in systemic and pulmonary infections
Since Histoplasma infection is primarily through the respiratory route, we also analyzed the IFN
-producing Vß populations in the lungs and the mediastinal lymph nodes. Mice were infected intratracheally and IFN
-producing cells were analyzed at day 14 after infection. Similar to what was observed in the spleens (Fig. 5), data in Figure 6 and Table 2 show that almost all Vß populations produced IFN
, except that some specific Vß populations constituted the major IFN
-producing populations. These included Vß3, Vß8.1&8.2, Vß13 and Vß17a of both CD4 and CD8 T cells and Vß5.1&5.2 specifically of CD8 T cells. It is worth noting that the major IFN
-producing cells in the lungs and MLN after intratracheal infection and that in the spleen after intravenously infection shared the same Vß usage (Table 2). The Vß3 and Vß8.1&8.2 subpopulations were major IFN
-producing CD4 T cells and the Vß3, Vß5.1&5.2 and Vß8.1&8.2 were major IFN
-producing CD8 T cells. Only Vß13 and Vß17a IFN
-producing CD4 and CD8 T cells were distinctly high in the lungs and MLN but not in the spleen. Thus, Histoplasma infection induces the expansion of a broad spectrum of Vß populations. While most expanded Vß populations are functionally active, some specific Vß populations of CD4 T cells and CD8 T cells are major IFN
producers.
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Discussion
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Histoplasma is an intracellular pathogen of the macrophages (5,26). The production of IFN
is thus critical to host protective immune response against histoplasmosis (1,5). However, the pool size of IFN
-producing cells in the host has never been determined. In this study, we utilized a single cell cytokine assay which can reliably enumerate the number of T cells that produce the cytokine (6,10,11,13), in order to quantify IFN
-producing cells. Our data showed that 8.4% of splenic CD4 T cells and 3.9% of CD8 T cells are actively engaged in IFN
production at the peak of response. The kinetics of response is similar for both CD4 and CD8 T cells. Since the whole yeast cell was used as stimulant, the responding cells measured are the sum of at least most of, if not all, antigen-experienced T cells that recognize different antigenic determinants. Although the magnitude of response to Histoplasma infection is not as impressive as that in the LCMV model, where 5070% of CD8 T cells are functionally active at the peak of infection, it is nevertheless a substantial response staged by the murine host to fight against histoplasmosis (12).
Recently, Pathan et al. used a single cell cytokine assay to identify the T cell population that contributes to protective immunity against tuberculosis and to evaluate the relationship between host response and disease (13). Individuals with a spectrum of clinical outcomes after contact with Mycobacterium tuberculosis were recruited for the study. Given the importance of CD4 T cells and IFN
in tuberculosis and the possible protective role of ESAT-6-specific T cells in vivo, they investigated whether the frequency of ESAT-6-specific IFN
-secreting CD4 T cells correlated with clinical outcome. Peripheral blood mononuclear cells obtained from different groups of patients and healthy controls were stimulated with ESAT-6 peptide. ELISPOT assay was used to quantify the frequency of EAST-specific IFN
-secreting cells. They found that healthy household contacts, and those with tuberculosis lymphadenitis, have higher frequencies of ESAT-6-specific IFN
-secreting T cells than those with pulmonary tuberculosis. The results of their study indicated that the frequency of ESAT-6-specific IFN
-secreting T cells correlated with the clinical disease state of the individual. The study also revealed that T cells specific for certain ESAT-6 epitopes are detected by ELISPOT assay but not by proliferation assay. These results showed that a functional assay detecting the cytokine that is important to host defense is a direct assessment of protective immunity and they established a direct relationship between the quantity of functional T cells and protection. Our study quantifying IFN
-producing cells in immune response to histoplasmosis in the murine host laid the groundwork for assessment of protective immune response in humans.
It is of interest to note in Figure 2 that rapid expansion of IFN
-producing cells (between days 7 and 14) occurred after the onset of fungal clearance. Unlike what was observed in the LCMV model where the onset of death phase of antigen-specific CD8 T cell starts after viral clearance (27,28), the death phase of IFN
-producing cells in the murine histoplasmosis model starts before complete fungal clearance. Bodovinac and Harty were the first to report a similar observation on the homeostatsis of CD8 T cells in mice infected with Listeria monocytogenes (29). Our observation, together with that made in the Listeria model, supports the concept that the regulation of T cell homeostasis is programmed and independent of the clearance of antigen (29). It is possible that the regulatory mechanism of T cell homeostasis in response to a virus differs from that to a non-viral pathogen. The onset of death phase of viral antigen-specific T cells occurs after complete viral clearance and the death of non-viral antigen-specific T cells is independent of antigen clearance. However, in murine histoplasmosis, although the initiation of death phase of functional T cells is not initiated by complete fungal clearance, their decline does coincide.
CD4 T cells are the major IFN
producers and are critical in host defense against histoplasmosis (1,5). The involvement of CD8 T cells in host defense has also been reported (3,30). Optimal fungal clearance requires CD8 T cells (3), and mice defective in perforin gene are susceptible to Histoplasma infection (30). Recently, the importance of CD8 T cells in vaccine-induced immunity against Histoplasma was reported (22). Repeated subcutaneous injections of live Histoplasma yeasts protected CD4 T cell-depleted mice from lethal intranasal challenge. In the same study, repeated subcutaneous injections of live Blastomyces also induced protection in mice against lethal challenge. CD8 T cells as well as CD4 T cells isolated from the lungs of mice vaccinated with live Blastomyces yeasts produced TNF
, IFN
and GM-CSF, implying that CD8 T cells like CD4 T cells are functional in mice vaccinated with Histoplasma (22). We demonstrated in this study that CD8 T cells in mice infected by Histoplasma, through either the intravenous or intratracheal route, are activated to produce IFN
(Fig. 1B). We also showed that CD4 and CD8 T cell responses to Histoplasma infection follow similar kinetics, although the magnitude of CD8 T cell response is lower than that of CD4 T cells (Fig. 2). As Histoplasma is an intracellular pathogen of the macrophage, our data showing that CD8 T cells produce IFN
contributes to the understanding of host defense against the fungus.
We selected three activation-related surface markers to study surface phenotype of functional T cells and showed that surface expression of CD44hi but not CD25 or O-glycosylated CD43 correlates with T cell IFN
production (Fig. 3). CD44 is a cell adhesion molecule belonging to the hyaluronate receptor family (31). The functional role of CD44hi expression on activated T cells has been explored but not resolved (3235). It has been shown that CD44 and hyaluronic acid interaction targets lymphocytes to extralymphoid organs (32), thus CD44 plays a role in controlling lymphocyte migration to the inflammatory sites. In M. tuberculosis pulmonary infection, mice with CD44 deficiency have a lower survival rate and enhanced mycobacterial growth in the lungs and livers (33). In addition, T cell and macrophage recruitment into the lungs is impaired and the concentration of IFN
is reduced in lung homogenate (33). This study showed that CD44 is important for the recruitment of T cells and macrophages, thus playing an important role in the protective immunity against pulmonary tuberculosis. On the contrary, in CD44-deficient mice infected with a low dose of mycobacteria, neither the bacterial load nor the migration of activated T cells to the inflamed lungs is affected (34). Therefore, CD44 expression is not necessary for trafficking of protective T cells to the lungs in low dose infection. A recent study by Blass et al. (35) suggests a regulatory role of CD44 in T cell production of IFN
. Toxoplasma gondii infection upregulates the active form of CD44 in CD4 T cells. Treating infected animals with anti-CD44 antibody abolishes the development of IFN
-dependent immunopathology. Adding anti-CD44 antibody to CD4 T cells from infected mice without the presence of complement reduces IFN
production (35). Our study showing a strong correlation between IFN
-production and CD44hi expression throughout the course of infection indicates that CD44hi phenotype is the best marker for T cell effector function, whether the role of CD44 is important for cytokine production or for cytokine-producing cells to traffic to the inflamed tissue or both. It is worth noting that not all cells that expressed CD44hi marker were IFN
producers. While most IFN
-producing cells had CD44hi phenotype, only about 1/3 of CD44hi cells produced IFN
. Therefore, expression of high numbers of CD44 molecule is necessary but not sufficient for functional T cells to produce IFN
.
In their pulmonary histoplasmosis model, Gomez et al. analyzed lymphocyte Vß usage in mice infected with Histoplasma (18,36). They obtained cDNA products by RTPCR method using Cß1 antisense primer to reversely transcribe RNA from lymphocytes of infected mice, and 20 different Vß-specific sense primers and a common Cß2 antisense primer to amplify cDNA. Comparing the relative quantity of each Vß-specific cDNA product, they demonstrated a significant increase of Vß4+ T cells in the lungs of mice with primary pulmonary histoplasmosis (18). Their study showed that the Vß repertoire is biased in the lungs of mice infected with Histoplasma, but the Vß population that produces IFN
was not studied (36). Depleting Vß4+ T cell population delayed clearance of the fungus, but the animals still resolved the infection (18). In the current study, we used anti-CD4, anti-CD8 antibodies, a panel of anti-Vß monoclonal antibodies and intracytoplasmic staining of IFN
to assess the percentage of each IFN
-producing Vß population in the spleen, lung and MLN of infected mice and demonstrated that Vß4+ T cells is not the only population that undergoes expansion. Instead, a broad spectrum of Vß populations expand and most of the expanded populations are functional in producing IFN
(Figs 5 and 6 and Table 2). Our data showing the expansion of multiple IFN
-producing Vß populations after infection can explain why in Gomez's study depletion of only the Vß4 population does not abolish protective immunity (18).
Furthermore, in mice infected intratracheally with Histoplasma, the major IFN
-producing T cells in the lungs and the draining lymph nodes shared the same Vß usage. These results indicate that a broad spectrum of Vß cells expand in the lymph node after infection and the whole spectrum, but not one specific Vß subpopulation of cells, are recruited to the lungs. Differences in methodology, genetic background of the animals, route of infection and strains of Histoplasma yeasts we used could account for the discrepancy of our findings.
Using staining and flow cytometry to study the expansion of specific Vß populations was different from that by RTPCR method. While the RTPCR method does not differentially amplify either CD4 or CD8 T cells, staining and flow cytometry can separately analyze each CD4+ and CD8+ T Vß cell subpopulation (37,38). In addition, multiple-color staining of intracytoplasmic IFN
, cell surface phenotypic marker and Vß subtype, identifies and quantifies the functional Vß subpopulations in the CD4+ and CD8+ T cell populations. Importantly, even a minor population would not be missed. The disadvantage of using staining to identify Vß populations is that it is limited by the availability of antibodies. Since there were no specific monoclonal antibodies to separately identify Vß5.1/Vß5.2 and Vß8.1/Vß8.2, the contribution of each of them could not be assessed.
Data in Table 2 showed that in mice infected intratracheally with Histoplasma, the major Vß subpopulations (Vß3, Vß8.1 &8.2, Vß13 and Vß17a of CD4 T cells and Vß3, Vß5.1&5.2, Vß8.1&8.2, Vß13 and Vß17a of CD8 T cells) of the IFN
-producing T cells in the lungs and the draining lymph nodes overlap with that in the spleens (Vß3 and Vß8.1&8.2 of CD4 T cells and Vß3, Vß5.1&5.2 and Vß8.1&8.2 of CD8 T cells) after systemic infection. While the route of infection may account for the expansion of different but overlapping Vß subpopulations in the lungs, MLN and the spleen, it does not explain the discrepancy of the findings of Gomez and ours. Since C57BL/6 mice from the same source were used in both of our studies, the genetic background of animals should not account for the discrepancy of our findings (18). However, it still remains to be determined whether infection with different strains of Histoplasma yeast cells could account for the discrepancy.
In this study, we characterized antigen-specific functional CD4 and CD8 T cells in the host after Histoplasma infection. We showed that not only CD4 T cells but also CD8 T cells are activated and engage in IFN
production. A strong correlation of CD44hi expression and IFN
production was observed in both CD4 and CD8 T cells throughout the course of infection. Furthermore, infection induces expansion of a broad spectrum of Vß populations, suggesting no TCR bias in primary immune response to histoplasmosis. Almost all expanded Vß populations in the spleen, lungs and the draining lymph nodes are functional in producing IFN
, although some have a greater contribution than others. Our study is the first to provide an overall understanding of the kinetics, the magnitude of response and the characteristics of the antigen-specific, functionally important CD4 and CD8 T cells in histoplasmosis.
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Acknowledgements
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This work was supported by funds from the Republic of China National Science Council Grants NSC 89-2320-B-002-195 and NSC 90-2320-B-002-139.
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Abbreviations
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IFN | interferon gamma |
MLN | mediastinal lymph nodes |
TCR | T cell receptor |
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Notes
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Transmitting editor: S. H. E. Kaufmann
Received 1 March 2004,
accepted 30 August 2004.
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