Diversity of the T-Cell Response to Pulmonary Cryptococcus neoformans
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《感染与免疫杂志》
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine
Immunology Graduate Program
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan
ABSTRACT
Cell-mediated immunity plays an important role in immunity to the pathogenic fungus Cryptococcus neoformans. However, the antigen specificity of the T-cell response to C. neoformans remains largely unknown. In this study, we used two approaches to determine the antigen specificity of the T-cell response to C. neoformans. We report here that a diverse T-cell receptor (TCR) V repertoire was maintained throughout the primary response to pulmonary C. neoformans infection in immunocompetent mice. CD4+ T-cell deficiency resulted in relative expansion of all CD8+ T-cell subsets. During a secondary immune response, preferential usage of a TCR V subset in CD4+ T cells occurred in single individuals, but the preferences were "private" and not shared between individuals. Both CD4+ and CD8+ T cells from the secondary lymphoid tissues of immunized mice proliferated in response to a variety of C. neoformans antigens, including heat-killed whole C. neoformans, culture filtrate antigen, C. neoformans lysate, and purified cryptococcal mannoprotein. CD4+ and CD8+ T cells from the secondary lymphoid tissues of mice undergoing a primary response to C. neoformans proliferated in response to C. neoformans lysate. In response to stimulation with C. neoformans lysate, lung CD4+ and CD8+ T cells produced the effector cytokines tumor necrosis factor alpha and gamma interferon. These results demonstrate that a diverse T-cell response is generated in response to pulmonary C. neoformans infection.
INTRODUCTION
The fungal pathogen Cryptococcus neoformans can infect immunocompetent individuals, but this organism causes disease primarily in individuals with defects in cellular immunity (5). T-cell-mediated immunity plays a critical role in clearance of pulmonary C. neoformans infection in mice, but the antigen specificity of the responding CD4+ and CD8+ T cells remains largely unknown. In this study, we used two approaches to determine the antigen specificity of the T-cell response to C. neoformans.
In T cells, which comprise the majority of T cells, the T-cell receptor (TCR) is expressed as a heterodimeric protein composed of and subunits. Somatic recombination of diversity and joining regions in V and somatic recombination of variable, diversity, and joining regions in V result in the diversity of the TCR repertoire (3). A number of studies, including studies of the pathogenic fungus Histoplasma capsulatum, have demonstrated that there are V preferences during T-cell responses, which correlate to T-cell function or antigen specificity (7-11). Similar to control of C. neoformans infection, control of H. capsulatum infection is primarily dependent on CD4+ T cells. Based on these results, we studied the V TCR expression by CD8+ and CD4+ T cells during pulmonary C. neoformans infection.
Our first objective in this study was to characterize the V TCR usage by CD4+ and CD8+ T cells during primary and secondary responses to pulmonary C. neoformans infection. Our goals were to determine (i) whether preferential expansion of specific V subsets by either T-cell subset occurs during primary infection; (ii) whether V skewing, if present, represents superantigen-induced proliferation or oligoclonal expansion of antigen-specific T cells; and (iii) whether tracking a single or limited number of V subsets throughout the course of the T-cell response could be used as a surrogate marker for antigen specificity.
C. neoformans has been shown to have mitogenic activity with human T cells in vitro (20, 21, 25). Superantigens are microbially derived proteins which bind directly to the V region of a limited number of closely related TCR V subsets (17). Neonatal exposure to mouse mammary tumor virus superantigens results in mouse strain-specific deletion of TCR V subsets from the mature TCR repertoire (1). Exposure of mature T cells to superantigens (such as staphylococcal enterotoxin B) results in rapid expansion of T cells bearing V bound by the superantigen, followed by activation-induced cell death. Our second objective was to determine whether C. neoformans had superantigen activity in mice (in vitro or in vivo). In addition, we sought to determine the proliferative and effector cytokine responses of CD4+ and CD8+ T cells from the lungs and the secondary lymphoid tissues in response to C. neoformans antigens.
MATERIALS AND METHODS
Mice. Female CBA/J mice (weight, 25 ± 4 g; age, 5 to 7 weeks) were obtained from the Jackson Laboratories (Bar Harbor, Maine). The mice were housed under pathogen-free conditions in enclosed filter-topped cages. Clean food and water were provided ad libitum. The mice were handled and maintained using microisolator techniques, and there was daily veterinarian monitoring. Bedding from the mice was transferred weekly to cages of uninfected sentinel mice that were subsequently bled at weekly intervals and were found to be negative for antibodies to mouse hepatitis virus, Sendai virus, and Mycoplasma pulmonis. All studies involving mice were approved by the University Committee on Use and Care of Animals at the University of Michigan.
C. neoformans. C. neoformans strain 52D (= ATCC 24067) was obtained from the American Type Culture Collection (Manassas, VA). For infection, the yeast was grown to the stationary phase (48 to 72 h) at 35°C in Sabouraud dextrose broth (1% neopeptone and 2% dextrose; Difco, Detroit, Mich.) on a shaker. The cultures were then washed in nonpyrogenic saline, counted with a hemacytometer, and diluted to obtain a concentration of 3.3 x 105 CFU/ml in sterile nonpyrogenic saline. The precise number of organisms delivered was determined by counting the CFU in an inoculum plated on Sabaraud dextrose agar (Difco).
Intratracheal inoculation of C. neoformans. Mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg; Fort Dodge Laboratories, Fort Dodge, IA) and xylazine (6.8 mg/kg; Lloyd Laboratories, Shenandoah, IA) and were restrained on a small surgical board. A small incision was made through the skin over the trachea, and the underlying tissue was separated. A 30-gauge needle was attached to a 1-ml tuberculin syringe filled with a diluted C. neoformans culture. The needle was inserted into the trachea, and 30 μl of inoculum (104 CFU) was dispensed into the lungs. The needle was removed, and the skin closed with cyanoacrylate adhesive. The mice recovered with minimal visible trauma.
Lung, lymph node, and spleen leukocyte isolation. The lungs of each mouse were excised, washed in phosphate-buffered saline (PBS), minced, and digested enzymatically for 30 min in 15 ml/lung of digestion buffer (RPMI, 5% fetal calf serum [FCS], 1 mg/ml collagenase [Boehringer Mannheim Biochemical, Chicago, IL], 30 μg/ml DNase [Sigma Chemical Co., St. Louis, MO]). Following erythrocyte lysis using NH4Cl buffer, the cells were washed, resuspended in complete medium, and centrifuged for 30 min at 2,000 x g in the presence of 20% Percoll (Sigma) to separate leukocytes from the cell debris and epithelial cells. Total numbers of lung leukocytes were determined in the presence of trypan blue using a hemocytometer; the viability was >85%. Lung-associated lymph nodes (LALN) and spleens were excised, and the cells dispersed with the plunger of a 3-ml syringe. Erythrocytes were lysed using NH4Cl buffer, and the cells were resuspended in complete medium (RPMI, 5% FCS, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin sulfate).
Flow cytometry. For surface staining, leukocytes were washed and resuspended at a concentration of 107 cells/ml in FA buffer (Difco, Detroit, MI) containing 0.1% NaN3 (Sigma), and Fc receptors were blocked by addition of anti-CD16/32 (Fc block; BD Pharmingen, San Diego, CA). Following Fc receptor blocking, 106 cells were stained in 120-μl (final volume) mixtures in polystyrene tubes (12 by 75 mm; BD Pharmingen) for 20 min at 4°C. Leukocytes were stained with the following antibodies, according to the manufacturer's instructions: CD4 (RM4-4 and H129.19), CD8 (5H10-1), CD8 (53-5.8), and a mouse TCR V screening kit (BD Pharmingen). Preliminary experiments confirmed that CBA/J mice did not express V 3, 5, 6, 7, 8.1, 9, 12, and 17 due to expression of endogenous proviruses and mouse mammary tumor viruses and were thus were not included in the V analysis. Cells were washed twice with FA buffer and resuspended, and 200 μl of 4% formalin (Fisher Chemical, Pittsburgh, PA) was added to fix the cells. A minimum of 20,000 events were acquired with a FACScaliber flow cytometer (BD Pharmingen) using the Cell-Quest software (BD Pharmingen).
Intracellular flow cytometry. Leukocytes (2 x 106 cells/ml) were cultured for 12 h in 12-well plates in the presence of 0.1 μg/ml soluble anti-CD3 (145-2C11; BD Pharmingen) with or without 0.1 μg/ml anti-CD28 (37.51; BD Pharmingen). Brefeldin A and/or monensin (in the form of Golgi-stop or Golgi-block) were added for the last 4 h of incubation according to the manufacturer's instructions (BD Pharmingen). Nonadherent cells were harvested and washed twice with FA buffer, and staining for cell surface molecules was performed as described above. For intracellular staining, cells were washed to remove excess surface stains, fixed and permeabilized using Cytofix/Cytoperm (BD Pharmingen), and stained using anti-gamma interferon (IFN-) (XMG1.2) or anti-tumor necrosis factor alpha (TNF-) (MP6-XT22; BD Pharmingen) in permeabilization buffer (FA buffer containing 0.1% saponin [Sigma]) at 4°C for 30 min. Flow cytometry was performed as described above for surface staining, except that >50,000 events per sample were routinely collected. The specificity of anticytokine antibodies was tested by comparing staining of experimental samples to at least two of the following three negative controls: (i) isotype control, (ii) excess unlabeled antibody, and (iii) preincubation of antibody with recombinant cytokine.
In some experiments, T cells were enriched from the lungs by fluorescence-activated cell sorting (FACS) and from lymph nodes and spleens by magnetism-activated cell sorting (MACS). For FACS, lung leukocytes were stained using anti-CD4 (RM4-4) and anti-CD8 (5H10-1). Cell sorting was performed at the University of Michigan Cancer Center Flow Cytometry Core with a FACSVantage SE cell sorter (BD Immunocytometry Systems, San Jose, CA). The purity of the sorted population was >99%, as determined by postsorting analysis. For MACS, cell suspensions from secondary lymphoid tissues were stained using a panel of biotinylated antibodies, including anti-CD19 (1D3), anti-CD49b (DX5), anti-Gr-1 (RB6-8C5), and anti-erythroid cells (TER-119) (all obtained from BD Pharmingen), as well as anti-mouse F4/80 (CI:A3-1; Caltag Laboratories, Burlingame, CA), and T cells were enriched by negative selection using antibiotin microbeads with a SuperMACS separator (Miltenyi Biotec, Auburn, CA). A total of 106 enriched T cells were cocultured with 106 adherent lung cells from uninfected mice and stimulated with either (i) anti-CD3 and anti-CD28 or (ii) C. neoformans lysate.
Proliferation assay. Cells were assayed for proliferation using an in vitro fluorescence-based assay. Briefly, 2 x 106 cells from the various organs were stained with 5 μM 5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) in PBS containing 5% FCS for 7 min at room temperature. The cells were washed three times to remove the excess CFSE and cultured for 3 days in the presence or absence of anti-CD3 antibodies (0.1 μg/ml). A minimum of 20,000 events were acquired with a FACScaliber flow cytometer (BD Pharmingen) using the Cell-Quest software (BD Pharmingen).
T-cell depletion using monoclonal antibodies. Depletion of CD4+ T cells was accomplished by intraperitoneal administration of monoclonal antibodies. Anti-CD4 (GK1.5, rat immunoglobulin G2b) was prepared from ascites by dilution in nonpyrogenic saline and filtration through a 0.45-μm syringe filter. Mice received 200 μg of GK1.5 or a nonpyrogenic saline control in 200 μl nonpyrogenic saline at zero time and on day 1 of infection, followed by 200 μg every 8 days. The efficiency of T-cell depletion was assessed by flow cytometric analysis using anti-CD4 (RM 4-4), which binds a region of CD4 distinct from the region bound by GK1.5. The efficiencies of CD4+ T-cell depletion in the lungs (>98%) and spleens (>99%) of mice were calculated by comparing the numbers of T cells in treated mice with the numbers of T cells in controls.
Preparation of C. neoformans antigens. (i) Heat-killed C. neoformans. C. neoformans strain 52D (= ATCC 24067) was obtained from the American Type Culture Collection. This yeast was grown to the stationary phase (72 h) at 34°C in Sabouraud dextrose broth (1% neopeptone and 2% dextrose; Difco, Detroit, Mich.) on a shaker platform. The yeast was washed three times in phosphate-buffered saline, counted with a hemacytometer, resuspended at a concentration of 109 cells/ml, and incubated in a 60°C water bath for 2 h with periodic mixing. The absence of viable organisms was confirmed by plating an aliquot containing 108 organisms on Sabouraud dextrose agar (Difco, Detroit, MI).
(ii) C. neoformans culture filtrate. C. neoformans culture filtrate (CneF) was prepared from C. neoformans grown to the stationary phase in asparagine broth. The yeast cells were pelleted by centrifugation at 1,500 x g for 30 min. The supernatant was filtered though a 0.22-μm bottle top filter (Corning Incorporated, Corning, NY). The filtered culture supernatant was concentrated 10-fold using an Amicon concentration apparatus with a 50-kDa-cutoff filter (Millipore, Billerica, MA) and then was washed with five times the original culture volume of nonpyrogenic saline. The protein concentration of CneF (622 μg/ml) was determined by the bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL).
(iii) Purified C. neoformans mannoprotein. Mannoprotein purified from culture supernatants of acapsular C. neoformans strain ATCC 52817 (19, 22) was a generous gift from Stuart Levitz, Boston University.
(iv) C. neoformans lysate. Lysate was prepared from stationary-phase organisms as follows. A 30-ml yeast culture was centrifuged at 1,500 x g for 20 min and washed three times with PBS, and the cells were resuspended in a minimal volume of PBS. Then 0.5-mm glass beads were added to cover the yeast (Biospec Products, Bartlesville, OK). The yeast cells were lysed by 25 cycles consisting of 30 s of vortexing and 30 s in an ice bath. The yeast lysate was diluted with 10 ml PBS and separated from the cellular debris by low-speed centrifugation (750 x g), and the supernatant was filtered through a 0.22-μm bottle top filter. The sterile lysate was frozen at –70°C until it was used.
Secondary pulmonary C. neoformans infection in mice. CBA/J mice were infected intratracheally with 103 CFU of C. neoformans strain 52D. The mice were housed in specific-pathogen-free conditions with food and water provided ad libitum for 16 weeks to allow clearance of the primary infection. For secondary infections, mice were then infected intratracheally with 104 CFU C. neoformans (a dose that was 10-fold higher than the primary dose).
Statistics. For preferential V usage during primary infection in C. neoformans-infected CBA/J mice, an analysis of variance was used to compare fold increases in each V+ T-cell population. A P value of <0.05 was considered statistically significant.
RESULTS
V TCR repertoires in the spleens and lungs of naive CBA/J mice. In order to establish a baseline to determine the relative enrichment of any particular V subset, it was necessary to first determine the naive V repertoire in uninfected control mice. To do this, uninfected specific-pathogen-free mice were sacrificed, and leukocytes were recovered from spleens as described in Materials and Methods. The expression of various V subsets was determined by flow cytometry using a commercially available screening kit. The V repertoires in the spleens were different for CD4+ and CD8+ T cells. While some V subsets were expressed at higher frequencies in both CD4+ and CD8+ T cells (V 8.2 and V 8.3) (Fig. 1A and B), other V subsets were expressed differentially in the populations. V 2, 11, 13, and 14 were expressed at higher frequency in CD8+ T cells, and V 4 and 10 were expressed at higher frequencies in CD4+ T cells (Fig. 1A and B). Although V 11-expressing T-cell subsets would be expected to be eliminated from CBA/J mice, based on their expression of mammary tumor virus 9, incomplete deletion of the V 11+ CD8+ subset has been described previously (16). In the CD4+ or CD8+ compartment, however, the relative frequency of each V subset was relatively conserved (Fig. 1A and B). Thus, the splenic TCR V repertoire was conserved from one animal to another in both CD4+ and CD8+ T cells.
Our ultimate goal was to identify populations of T cells that were enriched during pulmonary C. neoformans infection. To determine whether the population of T cells in the lungs mirrored the population in the spleens of uninfected mice, we determined the frequency of each V subset expressed by CD4+ and CD8+ T cells from the lungs of uninfected mice. In the CD8+ T-cell compartment, there was slightly more variability in the TCR V usage of CD8+ T cells from the lungs than in the TCR V usage of CD8+ T cells from the spleens (Fig. 1A and C). Lung CD4+ T cells had a pattern of V usage similar to that of CD4+ T cells from the spleen (Fig. 1B and D). The TCR V usage of CD4+ T cells from the lungs was conserved, similar to the results found for lung CD8+ T cells, but the frequencies of some CD4+ TCR V subsets were more variable in the lungs than in the spleen (Fig. 1B and D). The total numbers of CD4+ and CD8+ T cells in the lungs were consistent for five uninfected mice, and so the absolute number of each V subset mirrored the frequencies (Fig. 1E and F). Thus, although the V TCR repertoire in lung T cells was similar to that in the splenic compartment and was conserved, there was some heterogeneity in the V TCR repertoire in lung T cells.
Lung TCR V repertoire during pulmonary C. neoformans infection. Our next objective was to determine whether particular V subsets were enriched in either the CD4+ or CD8+ T-cell populations during pulmonary C. neoformans infection. In response to pulmonary C. neoformans infection, resistant mice recruit both CD4+ and CD8+ T cells to the lungs (13). CD4+ T cells are relatively enriched, particularly late in the response (13). To control for differences in the number of T cells in the lungs of infected mice, we converted data to absolute numbers of each V subset per lung and determined the relative increase compared to uninfected controls for each subset. The absolute number and fold increase for CD4+ and CD8+ T cells expressing each of the V subsets were determined for mice at weeks 1, 3, and 5 postinfection. At week 1 postinfection, no V subsets were increased by more than onefold (data not shown), and there was no difference between subsets. At the peak of the T-cell response (week 3 postinfection), a modest increase in each of the CD8+ T-cell V subsets was observed, which represented 2.7- to 4.3-fold expansion compared to uninfected controls (Fig. 2D). Similarly, in CD4+ T cells, expansion of all V subsets occurred in response to infection (Fig. 2B). In contrast to the data for CD8+ T cells, the CD4+ V subsets were all expanded more than 11-fold compared to the CD4+ V subsets for uninfected control lungs (Fig. 2B). The number of V13+ CD4+ T cells was significantly expanded compared to the controls (>70-fold) (Fig. 2B); however, V13+ CD4+ T cells comprised a minority of the total CD4+ T-cell population in the lungs (6.3%) (Fig. 2A). This preferential expansion of V13+ CD4+ T cells was not observed at week 5 postinfection (Fig. 2F), and V13+ T cells were an even smaller proportion of the total lung CD4+ T cells (1.9%). Thus, during primary pulmonary C. neoformans infection, a diverse TCR V repertoire was maintained by both CD4+ and CD8+ T cells infiltrating the lungs.
Lung CD8+ TCR V repertoire during pulmonary C. neoformans infection in CD4+ T-cell-deficient mice. Pulmonary C. neoformans infection primarily affects individuals with compromised CD4+ T-cell immunity (5). To determine the effect of CD4+ T-cell deficiency on the resulting CD8+ TCR V repertoire, mice were made CD4+ T cell deficient by systemic administration of anti-CD4 depleting monoclonal antibodies as described in Materials and Methods. The expansion of CD8+ T cells belonging to the various TCR V subsets was assayed as described above. At week 3 postinfection, higher numbers of CD8+ T cells in each of the V subsets were present in the lungs compared to the numbers in CD4+ mice (Fig. 3A and 2D). The numbers represented a much greater expansion compared to uninfected controls (range, 6.9- to 42.0-fold), and V 2, 4, 10, and 14 all expanded more than 25-fold (Fig. 3A). At week 5 postinfection, V 2, 4, and 10 were expanded more than 35-fold (Fig. 3B). These numbers reflect total CD8+ T-cell expansion, which was exaggerated in the absence of CD4+ T cells. Thus, CD4+ T-cell deficiency resulted in expansion of all CD8+ V subsets compared to CD4+ mice, but the expansion was particularly pronounced for V subsets 2, 4, and 10.
TCR V repertoire during the secondary immune response to pulmonary C. neoformans infection. To determine whether mice maintained a diverse T-cell V repertoire during secondary infection, mice were infected with 103 CFU of C. neoformans and then housed in specific-pathogen-free conditions for 16 weeks to allow resolution of this primary infection. The mice were then given a secondary challenge consisting of 104 CFU, and the V repertoire was characterized 10 days after the secondary challenge. In the CD8+ T-cell compartment, very little expansion of CD8+ T cells was observed, and four of eight V subsets were contracted compared to uninfected controls (Fig. 4A). Whether increased numbers in the other four V subsets represented skewing of the CD8+ V repertoire in the secondary response is unclear, because the total number of CD8+ T cells at day 10 after the secondary challenge was similar to the number of cells in uninfected controls (data not shown). In the CD4+ T-cell compartment, preferential expansion of some V subsets was observed in individuals (Fig. 4B). For example, mouse 4 exhibited skewing of the CD4+ repertoire to V 4 and V 13, whereas mouse 3 preferentially expanded V 8.3 and V 14 (Fig. 4B). However, preferential usage of V subsets by CD4+ T cells was not consistent between individuals. Thus, during the secondary T-cell response to pulmonary C. neoformans infection, individuals exhibited skewing of the CD4+ T-cell repertoire, but consistent, preferential expansion of any V subset was not observed across the group.
Proliferative responses of T cells from immunized mice in response to C. neoformans antigens. The results described above demonstrated that a diverse TCR V repertoire was maintained in the T-cell response to pulmonary C. neoformans infection. Additionally, these results suggested that tracking any V subset would likely be a poor surrogate for antigen specificity in the T-cell response to pulmonary C. neoformans infection. Therefore, we shifted the focus of our investigation to determining whether we could identify a method for more specifically assaying the T-cell responses to C. neoformans.
To do this, we studied the proliferative responses of CD4+ and CD8+ T cells from mice immunized with C. neoformans. CBA/J mice were infected with 103 CFU of C. neoformans and allowed to recover from the infection. Previous studies demonstrated that there were enhanced T-cell responses to secondary C. neoformans infection in such mice (D. Lindell, M. Ballinger, R. McDonald, G. Towes, and G. Huffnagle, submitted for publication). At 16 weeks postinfection, single-cell suspensions were prepared from pooled lymph nodes and spleens of immunized mice. To assay for C. neoformans-specific responses, we generated a variety of antigen preparations from C. neoformans, as described in Materials and Methods. C. neoformans can persist in the lungs of resistant mice long after a primary infection (Lindell et al., submitted). The levels of cells recovered from the secondary lymphoid organs of mice immune to C. neoformans mice were below the detectable levels of viable yeast cells (<50 CFU in the pooled sample for five mice).
The proliferation of CD4+ and CD8+ T cells from mice at week 16 postinfection was assessed using a flow cytometry-based proliferation assay, as described in Materials and Methods. This assay has an advantage over [3H]thymidine incorporation assays because the proliferation of multiple cell populations can be determined independently in a single treatment well. Following 2 days in culture, there was minimal proliferation (as indicated by a decrease in CFSE staining) of either CD4+ or CD8+ T cells from immunized mice in the absence of stimulation (Fig. 5). In response to anti-CD3/anti-CD28 restimulation, a majority of CD4+ and CD8+ T cells proliferated (Fig. 5). A slight decrease in CFSE intensity was observed in both CD4+ and CD8+ T cells stimulated with C. neoformans lysate (Fig. 5). In contrast, after 4 days in culture, significant proportions of both CD4+ and CD8+ T cells from immunized mice had proliferated in response to C. neoformans lysate (Fig. 5).
Proliferative responses by both CD4+ and CD8+ T cells were also observed in response to a number of other C. neoformans-derived antigen preparations, including heat-killed organisms, culture filtrate antigen (CneF), and purified cryptococcal mannoprotein (Fig. 6A). These results demonstrated that a portion of CD4+ and CD8+ T cells in the secondary lymphoid tissues of immune mice proliferate in response to a number of C. neoformans antigen preparations.
Effector cytokine production by T cells from immunized mice in response to C. neoformans antigens. Our next objective was to determine whether the preparations of C. neoformans antigens (heat-killed organisms, lysate, CneF, or mannoprotein) could be used to assay effector cytokine production. To do this, lymphocyte suspensions from immune mice were cultured with uninfected adherent splenocytes in the presence of each stimulus, and the production of IFN- and TNF- was assayed by intracellular flow cytometry, as described in Materials and Methods. In response to each of the antigen preparations, CD4+ T cells produced both IFN- and TNF- (Fig. 6B). In contrast, CD8+ T cells produced only IFN- in response to C. neoformans antigens (Fig. 6C). These results demonstrate that T cells from the secondary lymphoid tissues of immune mice produced effector cytokines in response to nonviable C. neoformans antigen preparations.
Proliferation of CD4+ and CD8+ T cells from LALN of mice with pulmonary cryptococcal infections. Our next objective was to determine whether T cells from the LALN of mice during primary pulmonary infection proliferate in response to C. neoformans antigens. To do this, we assayed proliferation of LALN CD4+ and CD8+ T cells in response to C. neoformans lysate. The results obtained with immune mice suggested that maximal proliferation and effector cytokine production could be assayed using lysate, relative to the other antigen preparations (Fig. 6). Adherent lung cells from uninfected mice were cultured with LALN cells from mice 2 weeks after a primary infection, as described in Materials and Methods. Minimal proliferation was observed when LALN T cells were not stimulated; however, LALN T cells from infected mice proliferated in response to C. neoformans lysate (Fig. 7). We also investigated whether C. neoformans lysate induced proliferation from naive T cells. Although no such activity has been demonstrated for murine T cells, components of the C. neoformans cell wall have been shown to have mitogenic activity with human T cells (21, 25). After 4 days in culture, there was no difference in CFSE staining between unstimulated T cells from uninfected spleens and T cells stimulated with C. neoformans lysate (Fig. 7). T cells from uninfected spleens were capable of proliferation, however, and divided extensively in response to anti-CD3/anti-CD28 stimulation (Fig. 7). Both CD4+ and CD8+ T cells obtained from the LALN of mice at week 2 postinfection proliferated in response to C. neoformans lysate (Fig. 7). These results demonstrated that C. neoformans lysate did not induce mitogenic proliferation in naive murine T cells and that both CD4+ and CD8+ T cells recovered from the LALN proliferated in response to C. neoformans lysate. These data support the conclusion that a portion of the T cells in the LALN during pulmonary C. neoformans infection are antigen specific.
Effector cytokine production by CD4+ and CD8+ T cells from C. neoformans-infected mice. Our next objective was to determine the frequencies of CD4+ and CD8+ T cells producing the effector cytokines IFN- and TNF- in the lungs and secondary lymphoid tissues of C. neoformans-infected mice. To do this, T cells from infected lungs, LALN, spleens, uninfected lungs, and uninfected spleens were enriched by MACS or FACS, as described in Materials and Methods. T cells were cultured with adherent cells from uninfected control lungs in the presence of no stimulus or C. neoformans lysate. The results were expressed as the increase in the number of cytokine-positive cells compared with the number in unstimulated samples.
CD4+ T cells from infected lungs, but not CD4+ T cells from secondary lymphoid tissues, produced IFN- at a high frequency in response to CD3/CD28 restimulation (Fig. 8). CD4+ T cells in infected lungs produced TNF- in response to CD3/CD28 restimulation, but the frequency of TNF-+ CD4+ T cells in uninfected tissues was equal to or higher than the frequency of TNF-+ CD4+ T cells in infected lungs (Fig. 8). These results demonstrated that in response to CD3/CD28 restimulation, lung CD4+ T cells from infected mice produced IFN-. CD4+ T cells regulated the production of TNF- less stringently than they regulated the production of IFN-, however, as CD3/CD28 stimulation resulted in production of TNF- by CD4+ T cells from uninfected mice. CD8+ T cells from lungs and secondary lymphoid tissues produced IFN- in response to CD3/CD28 restimulation (Fig. 8). However, similar frequencies of CD8+ T cells from uninfected tissues produced IFN- in response to CD3/CD28 (Fig. 8). Thus, CD8+ T cells can produce TNF- and IFN- in response to low-dose CD3/CD28 restimulation in the absence of infection.
In response to C. neoformans lysate, CD4+ T cells from secondary lymphoid tissues did not produce IFN- or TNF-. Markedly higher frequencies of IFN- and TNF- production were observed with CD4+ T cells from infected lungs (Fig. 8). Although anti-CD3/anti-CD28 restimulation resulted in significant IFN- production by CD8+ T cells from the secondary lymphoid tissues (Fig. 8), CD8+ T cells from secondary lymphoid tissues restimulated with lysate were poor effector cytokine producers (Fig. 8). Similar to the results obtained with CD4+ T cells, only lung CD8+ T cells from infected lungs produced IFN- and TNF- in response to C. neoformans lysate (Fig. 8). These data show that C. neoformans lysate induces minimal effector cytokine production by T cells in uninfected mice. Furthermore, these results demonstrate that lung T cells from C. neoformans-infected mice produce effector cytokines in response to in vitro restimulation with C. neoformans lysate and that effector cytokine production occurs at a higher frequency than it occurs in T cells from secondary lymphoid tissues.
DISCUSSION
In this paper we report that in immunocompetent mice, a diverse TCR V repertoire is maintained throughout the primary response to pulmonary C. neoformans infection. While V 13 CD4+ T cells were preferentially expanded at week 3 postinfection, they comprised a minority of the overall lung CD4+ T-cell population. Furthermore, this preferential expansion did not persist until week 5 postinfection. CD4+ T-cell deficiency resulted in relative expansion of all CD8+ T-cell subsets. During the secondary immune response, preferential usage of CD4+ T-cell V subsets occurred in individuals, but not overall. Both CD4+ and CD8+ T cells from the secondary lymphoid tissues of immunized mice proliferated in response to a variety of C. neoformans antigens, including heat-killed whole C. neoformans, culture filtrate antigen (CneF), C. neoformans lysate, and purified cryptococcal mannoprotein. CD4+ and CD8+ T cells from the secondary lymphoid tissues of immune mice produced IFN- in response to these stimuli, and CD4+ T cells also produced TNF-. Both CD4+ and CD8+ T cells from the secondary lymphoid tissues of mice exhibiting a primary response to C. neoformans proliferated in response to C. neoformans lysate. In response to stimulation with C. neoformans lysate, lung CD4+ and CD8+ T cells produced the effector cytokines TNF- and IFN-.
Our studies demonstrated that a diverse TCR V repertoire is maintained throughout the primary response, but focusing of the TCR V repertoire occurred in CD4+ T-cell populations of individuals during the secondary immune response. Pulmonary infection of mice with H. capsulatum leads to significant skewing of the TCR V repertoire, as does protective vaccination with H. capsulatum heat shock protein 60 (9, 10, 23). Depletion of the preferentially expanded population in either case adversely affects clearance (9, 10, 23). Evidence obtained with other experimental systems, however, suggests that V preferences during primary infection are the exception rather than the rule. During primary Listeria monocytogenes infection, epitope-specific T cells have a diverse TCR V repertoire (4). In a secondary response, however, focusing of the V repertoire occurs in the responding antigen-specific T-cell populations (4). Similar to the results reported here, variability between genetically identical individuals was also observed (4). A T-cell response in which a common TCR V repertoire is found in all individuals has been termed a "public" repertoire, whereas a "private" repertoire is specific to an individual (6). In a number of recent studies workers have sought to determine the relative roles of private and public repertoires, chiefly in models of T-cell responses to viruses (14, 26). The CD8+ T-cell response to influenza in C57BL/6 mice is directed primarily against the immunodominant epitopes NP366 and PA224. The response to NP366 is characteristically public, whereas the response to PA224 is largely private (14, 15, 26). Although similar numbers of NP366- and PA224-specific T cells are generated in the primary response, the secondary response is dominated by the public NP366 response, suggesting that public specificity may play a more prominent role in secondary T-cell responses (2). However, private specificity may play a more important role in regulating differential responses to heterologous infections in genetically identical mice (15). Although in our study we did not utilize techniques such as spectratyping, the differences in V profiles between individual mice suggest that the CD4+ T-cell V repertoire during secondary C. neoformans infection could be characterized as largely private.
Our results indicated that during CD4+ T-cell deficiency, C. neoformans infection led to enhanced expansion of all CD8+ V subsets compared to the expansion observed with CD4+ T-cell sufficiency. Previous studies demonstrated that hyperexpansion of CD8+ T cells occurs in response to C. neoformans during CD4+ T-cell deficiency (18). The IFN--producing CD8+ T-cell effectors mediate a level of protection in a CD4+ T-cell-deficient host (18). Together, these results demonstrate that while the absence of CD4+ T cells increases the magnitude of the CD8+ T-cell response to C. neoformans infection, the CD8+ response to C. neoformans in a CD4+ T-cell-deficient host is also characterized by diversity.
If C. neoformans has superantigen activity in vivo in mice, we would have expected specific V subsets to be expanded in response to C. neoformans infection. Superantigens are microbially derived proteins which stimulate the proliferation of T cells in a non-antigen-specific manner via direct binding of the V region of the T-cell receptor (17). Superantigens may benefit a pathogen by inducing a high frequency of nonspecific T cells, which can interfere with the antigen-specific response (12). C. neoformans produces a mitogen (CnM) for human T cells, whose activity is localized to proteins found in the cell wall and membrane and can stimulate naive T-cell proliferation (20, 21). The mitogenic effect is similar to that of staphyloccocal enterotoxin B, a known superantigen, and requires phagocytosis and protein processing (24). In our study, we observed no proliferation by CD4+ or CD8+ T cells in response to C. neoformans lysate, even after 5 days in culture. Thus, our results suggest that C. neoformans mitogen is not active in mice.
Our data demonstrated that while T cells from the lymph nodes or spleens of mice at 2 weeks postinfection produced little TNF- and IFN- in response to stimulation with C. neoformans lysate, a significant number of T cells in the lungs produced each of these effector cytokines (Fig. 8). These results show that there is compartmentalization of T-cell effector function during pulmonary C. neoformans infection. T cells encounter antigen-presenting cells in the LALN bearing cognate antigen, where they respond by proliferating. Concomitant with trafficking to the lungs, both CD4+ and CD8+ T cells acquire an antigen-dependent effector cytokine-producing phenotype. Throughout the immune response to pulmonary C. neoformans infection, both CD4+ and CD8+ T cells maintain a diverse TCR V repertoire, and the response is driven by multiple C. neoformans antigens rather than by one or two immunodominant antigens.
ACKNOWLEDGMENTS
We thank Stuart Levitz and his laboratory for providing purified mannoprotein.
This work was supported by National Institutes of Health grants R01-HL065912 (to G.B.H.), R01-AI059201 (to G.B.H.), R01-HL051082 (to G.B.T.), and T32-AI07413 (to D.M.L.) and by a Department of Veterans Affairs merit grant (to G.B.T.).
FOOTNOTES
Corresponding author. Mailing address: Pulmonary and Critical Care Medicine, 6301 MSRB III, University of Michigan Medical Center, Ann Arbor, MI 48109-0642. Phone: (734) 936-9369. Fax: (734) 764-2655. E-mail: ghuff@umich.edu.
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Immunology Graduate Program
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan
ABSTRACT
Cell-mediated immunity plays an important role in immunity to the pathogenic fungus Cryptococcus neoformans. However, the antigen specificity of the T-cell response to C. neoformans remains largely unknown. In this study, we used two approaches to determine the antigen specificity of the T-cell response to C. neoformans. We report here that a diverse T-cell receptor (TCR) V repertoire was maintained throughout the primary response to pulmonary C. neoformans infection in immunocompetent mice. CD4+ T-cell deficiency resulted in relative expansion of all CD8+ T-cell subsets. During a secondary immune response, preferential usage of a TCR V subset in CD4+ T cells occurred in single individuals, but the preferences were "private" and not shared between individuals. Both CD4+ and CD8+ T cells from the secondary lymphoid tissues of immunized mice proliferated in response to a variety of C. neoformans antigens, including heat-killed whole C. neoformans, culture filtrate antigen, C. neoformans lysate, and purified cryptococcal mannoprotein. CD4+ and CD8+ T cells from the secondary lymphoid tissues of mice undergoing a primary response to C. neoformans proliferated in response to C. neoformans lysate. In response to stimulation with C. neoformans lysate, lung CD4+ and CD8+ T cells produced the effector cytokines tumor necrosis factor alpha and gamma interferon. These results demonstrate that a diverse T-cell response is generated in response to pulmonary C. neoformans infection.
INTRODUCTION
The fungal pathogen Cryptococcus neoformans can infect immunocompetent individuals, but this organism causes disease primarily in individuals with defects in cellular immunity (5). T-cell-mediated immunity plays a critical role in clearance of pulmonary C. neoformans infection in mice, but the antigen specificity of the responding CD4+ and CD8+ T cells remains largely unknown. In this study, we used two approaches to determine the antigen specificity of the T-cell response to C. neoformans.
In T cells, which comprise the majority of T cells, the T-cell receptor (TCR) is expressed as a heterodimeric protein composed of and subunits. Somatic recombination of diversity and joining regions in V and somatic recombination of variable, diversity, and joining regions in V result in the diversity of the TCR repertoire (3). A number of studies, including studies of the pathogenic fungus Histoplasma capsulatum, have demonstrated that there are V preferences during T-cell responses, which correlate to T-cell function or antigen specificity (7-11). Similar to control of C. neoformans infection, control of H. capsulatum infection is primarily dependent on CD4+ T cells. Based on these results, we studied the V TCR expression by CD8+ and CD4+ T cells during pulmonary C. neoformans infection.
Our first objective in this study was to characterize the V TCR usage by CD4+ and CD8+ T cells during primary and secondary responses to pulmonary C. neoformans infection. Our goals were to determine (i) whether preferential expansion of specific V subsets by either T-cell subset occurs during primary infection; (ii) whether V skewing, if present, represents superantigen-induced proliferation or oligoclonal expansion of antigen-specific T cells; and (iii) whether tracking a single or limited number of V subsets throughout the course of the T-cell response could be used as a surrogate marker for antigen specificity.
C. neoformans has been shown to have mitogenic activity with human T cells in vitro (20, 21, 25). Superantigens are microbially derived proteins which bind directly to the V region of a limited number of closely related TCR V subsets (17). Neonatal exposure to mouse mammary tumor virus superantigens results in mouse strain-specific deletion of TCR V subsets from the mature TCR repertoire (1). Exposure of mature T cells to superantigens (such as staphylococcal enterotoxin B) results in rapid expansion of T cells bearing V bound by the superantigen, followed by activation-induced cell death. Our second objective was to determine whether C. neoformans had superantigen activity in mice (in vitro or in vivo). In addition, we sought to determine the proliferative and effector cytokine responses of CD4+ and CD8+ T cells from the lungs and the secondary lymphoid tissues in response to C. neoformans antigens.
MATERIALS AND METHODS
Mice. Female CBA/J mice (weight, 25 ± 4 g; age, 5 to 7 weeks) were obtained from the Jackson Laboratories (Bar Harbor, Maine). The mice were housed under pathogen-free conditions in enclosed filter-topped cages. Clean food and water were provided ad libitum. The mice were handled and maintained using microisolator techniques, and there was daily veterinarian monitoring. Bedding from the mice was transferred weekly to cages of uninfected sentinel mice that were subsequently bled at weekly intervals and were found to be negative for antibodies to mouse hepatitis virus, Sendai virus, and Mycoplasma pulmonis. All studies involving mice were approved by the University Committee on Use and Care of Animals at the University of Michigan.
C. neoformans. C. neoformans strain 52D (= ATCC 24067) was obtained from the American Type Culture Collection (Manassas, VA). For infection, the yeast was grown to the stationary phase (48 to 72 h) at 35°C in Sabouraud dextrose broth (1% neopeptone and 2% dextrose; Difco, Detroit, Mich.) on a shaker. The cultures were then washed in nonpyrogenic saline, counted with a hemacytometer, and diluted to obtain a concentration of 3.3 x 105 CFU/ml in sterile nonpyrogenic saline. The precise number of organisms delivered was determined by counting the CFU in an inoculum plated on Sabaraud dextrose agar (Difco).
Intratracheal inoculation of C. neoformans. Mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg; Fort Dodge Laboratories, Fort Dodge, IA) and xylazine (6.8 mg/kg; Lloyd Laboratories, Shenandoah, IA) and were restrained on a small surgical board. A small incision was made through the skin over the trachea, and the underlying tissue was separated. A 30-gauge needle was attached to a 1-ml tuberculin syringe filled with a diluted C. neoformans culture. The needle was inserted into the trachea, and 30 μl of inoculum (104 CFU) was dispensed into the lungs. The needle was removed, and the skin closed with cyanoacrylate adhesive. The mice recovered with minimal visible trauma.
Lung, lymph node, and spleen leukocyte isolation. The lungs of each mouse were excised, washed in phosphate-buffered saline (PBS), minced, and digested enzymatically for 30 min in 15 ml/lung of digestion buffer (RPMI, 5% fetal calf serum [FCS], 1 mg/ml collagenase [Boehringer Mannheim Biochemical, Chicago, IL], 30 μg/ml DNase [Sigma Chemical Co., St. Louis, MO]). Following erythrocyte lysis using NH4Cl buffer, the cells were washed, resuspended in complete medium, and centrifuged for 30 min at 2,000 x g in the presence of 20% Percoll (Sigma) to separate leukocytes from the cell debris and epithelial cells. Total numbers of lung leukocytes were determined in the presence of trypan blue using a hemocytometer; the viability was >85%. Lung-associated lymph nodes (LALN) and spleens were excised, and the cells dispersed with the plunger of a 3-ml syringe. Erythrocytes were lysed using NH4Cl buffer, and the cells were resuspended in complete medium (RPMI, 5% FCS, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin sulfate).
Flow cytometry. For surface staining, leukocytes were washed and resuspended at a concentration of 107 cells/ml in FA buffer (Difco, Detroit, MI) containing 0.1% NaN3 (Sigma), and Fc receptors were blocked by addition of anti-CD16/32 (Fc block; BD Pharmingen, San Diego, CA). Following Fc receptor blocking, 106 cells were stained in 120-μl (final volume) mixtures in polystyrene tubes (12 by 75 mm; BD Pharmingen) for 20 min at 4°C. Leukocytes were stained with the following antibodies, according to the manufacturer's instructions: CD4 (RM4-4 and H129.19), CD8 (5H10-1), CD8 (53-5.8), and a mouse TCR V screening kit (BD Pharmingen). Preliminary experiments confirmed that CBA/J mice did not express V 3, 5, 6, 7, 8.1, 9, 12, and 17 due to expression of endogenous proviruses and mouse mammary tumor viruses and were thus were not included in the V analysis. Cells were washed twice with FA buffer and resuspended, and 200 μl of 4% formalin (Fisher Chemical, Pittsburgh, PA) was added to fix the cells. A minimum of 20,000 events were acquired with a FACScaliber flow cytometer (BD Pharmingen) using the Cell-Quest software (BD Pharmingen).
Intracellular flow cytometry. Leukocytes (2 x 106 cells/ml) were cultured for 12 h in 12-well plates in the presence of 0.1 μg/ml soluble anti-CD3 (145-2C11; BD Pharmingen) with or without 0.1 μg/ml anti-CD28 (37.51; BD Pharmingen). Brefeldin A and/or monensin (in the form of Golgi-stop or Golgi-block) were added for the last 4 h of incubation according to the manufacturer's instructions (BD Pharmingen). Nonadherent cells were harvested and washed twice with FA buffer, and staining for cell surface molecules was performed as described above. For intracellular staining, cells were washed to remove excess surface stains, fixed and permeabilized using Cytofix/Cytoperm (BD Pharmingen), and stained using anti-gamma interferon (IFN-) (XMG1.2) or anti-tumor necrosis factor alpha (TNF-) (MP6-XT22; BD Pharmingen) in permeabilization buffer (FA buffer containing 0.1% saponin [Sigma]) at 4°C for 30 min. Flow cytometry was performed as described above for surface staining, except that >50,000 events per sample were routinely collected. The specificity of anticytokine antibodies was tested by comparing staining of experimental samples to at least two of the following three negative controls: (i) isotype control, (ii) excess unlabeled antibody, and (iii) preincubation of antibody with recombinant cytokine.
In some experiments, T cells were enriched from the lungs by fluorescence-activated cell sorting (FACS) and from lymph nodes and spleens by magnetism-activated cell sorting (MACS). For FACS, lung leukocytes were stained using anti-CD4 (RM4-4) and anti-CD8 (5H10-1). Cell sorting was performed at the University of Michigan Cancer Center Flow Cytometry Core with a FACSVantage SE cell sorter (BD Immunocytometry Systems, San Jose, CA). The purity of the sorted population was >99%, as determined by postsorting analysis. For MACS, cell suspensions from secondary lymphoid tissues were stained using a panel of biotinylated antibodies, including anti-CD19 (1D3), anti-CD49b (DX5), anti-Gr-1 (RB6-8C5), and anti-erythroid cells (TER-119) (all obtained from BD Pharmingen), as well as anti-mouse F4/80 (CI:A3-1; Caltag Laboratories, Burlingame, CA), and T cells were enriched by negative selection using antibiotin microbeads with a SuperMACS separator (Miltenyi Biotec, Auburn, CA). A total of 106 enriched T cells were cocultured with 106 adherent lung cells from uninfected mice and stimulated with either (i) anti-CD3 and anti-CD28 or (ii) C. neoformans lysate.
Proliferation assay. Cells were assayed for proliferation using an in vitro fluorescence-based assay. Briefly, 2 x 106 cells from the various organs were stained with 5 μM 5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) in PBS containing 5% FCS for 7 min at room temperature. The cells were washed three times to remove the excess CFSE and cultured for 3 days in the presence or absence of anti-CD3 antibodies (0.1 μg/ml). A minimum of 20,000 events were acquired with a FACScaliber flow cytometer (BD Pharmingen) using the Cell-Quest software (BD Pharmingen).
T-cell depletion using monoclonal antibodies. Depletion of CD4+ T cells was accomplished by intraperitoneal administration of monoclonal antibodies. Anti-CD4 (GK1.5, rat immunoglobulin G2b) was prepared from ascites by dilution in nonpyrogenic saline and filtration through a 0.45-μm syringe filter. Mice received 200 μg of GK1.5 or a nonpyrogenic saline control in 200 μl nonpyrogenic saline at zero time and on day 1 of infection, followed by 200 μg every 8 days. The efficiency of T-cell depletion was assessed by flow cytometric analysis using anti-CD4 (RM 4-4), which binds a region of CD4 distinct from the region bound by GK1.5. The efficiencies of CD4+ T-cell depletion in the lungs (>98%) and spleens (>99%) of mice were calculated by comparing the numbers of T cells in treated mice with the numbers of T cells in controls.
Preparation of C. neoformans antigens. (i) Heat-killed C. neoformans. C. neoformans strain 52D (= ATCC 24067) was obtained from the American Type Culture Collection. This yeast was grown to the stationary phase (72 h) at 34°C in Sabouraud dextrose broth (1% neopeptone and 2% dextrose; Difco, Detroit, Mich.) on a shaker platform. The yeast was washed three times in phosphate-buffered saline, counted with a hemacytometer, resuspended at a concentration of 109 cells/ml, and incubated in a 60°C water bath for 2 h with periodic mixing. The absence of viable organisms was confirmed by plating an aliquot containing 108 organisms on Sabouraud dextrose agar (Difco, Detroit, MI).
(ii) C. neoformans culture filtrate. C. neoformans culture filtrate (CneF) was prepared from C. neoformans grown to the stationary phase in asparagine broth. The yeast cells were pelleted by centrifugation at 1,500 x g for 30 min. The supernatant was filtered though a 0.22-μm bottle top filter (Corning Incorporated, Corning, NY). The filtered culture supernatant was concentrated 10-fold using an Amicon concentration apparatus with a 50-kDa-cutoff filter (Millipore, Billerica, MA) and then was washed with five times the original culture volume of nonpyrogenic saline. The protein concentration of CneF (622 μg/ml) was determined by the bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL).
(iii) Purified C. neoformans mannoprotein. Mannoprotein purified from culture supernatants of acapsular C. neoformans strain ATCC 52817 (19, 22) was a generous gift from Stuart Levitz, Boston University.
(iv) C. neoformans lysate. Lysate was prepared from stationary-phase organisms as follows. A 30-ml yeast culture was centrifuged at 1,500 x g for 20 min and washed three times with PBS, and the cells were resuspended in a minimal volume of PBS. Then 0.5-mm glass beads were added to cover the yeast (Biospec Products, Bartlesville, OK). The yeast cells were lysed by 25 cycles consisting of 30 s of vortexing and 30 s in an ice bath. The yeast lysate was diluted with 10 ml PBS and separated from the cellular debris by low-speed centrifugation (750 x g), and the supernatant was filtered through a 0.22-μm bottle top filter. The sterile lysate was frozen at –70°C until it was used.
Secondary pulmonary C. neoformans infection in mice. CBA/J mice were infected intratracheally with 103 CFU of C. neoformans strain 52D. The mice were housed in specific-pathogen-free conditions with food and water provided ad libitum for 16 weeks to allow clearance of the primary infection. For secondary infections, mice were then infected intratracheally with 104 CFU C. neoformans (a dose that was 10-fold higher than the primary dose).
Statistics. For preferential V usage during primary infection in C. neoformans-infected CBA/J mice, an analysis of variance was used to compare fold increases in each V+ T-cell population. A P value of <0.05 was considered statistically significant.
RESULTS
V TCR repertoires in the spleens and lungs of naive CBA/J mice. In order to establish a baseline to determine the relative enrichment of any particular V subset, it was necessary to first determine the naive V repertoire in uninfected control mice. To do this, uninfected specific-pathogen-free mice were sacrificed, and leukocytes were recovered from spleens as described in Materials and Methods. The expression of various V subsets was determined by flow cytometry using a commercially available screening kit. The V repertoires in the spleens were different for CD4+ and CD8+ T cells. While some V subsets were expressed at higher frequencies in both CD4+ and CD8+ T cells (V 8.2 and V 8.3) (Fig. 1A and B), other V subsets were expressed differentially in the populations. V 2, 11, 13, and 14 were expressed at higher frequency in CD8+ T cells, and V 4 and 10 were expressed at higher frequencies in CD4+ T cells (Fig. 1A and B). Although V 11-expressing T-cell subsets would be expected to be eliminated from CBA/J mice, based on their expression of mammary tumor virus 9, incomplete deletion of the V 11+ CD8+ subset has been described previously (16). In the CD4+ or CD8+ compartment, however, the relative frequency of each V subset was relatively conserved (Fig. 1A and B). Thus, the splenic TCR V repertoire was conserved from one animal to another in both CD4+ and CD8+ T cells.
Our ultimate goal was to identify populations of T cells that were enriched during pulmonary C. neoformans infection. To determine whether the population of T cells in the lungs mirrored the population in the spleens of uninfected mice, we determined the frequency of each V subset expressed by CD4+ and CD8+ T cells from the lungs of uninfected mice. In the CD8+ T-cell compartment, there was slightly more variability in the TCR V usage of CD8+ T cells from the lungs than in the TCR V usage of CD8+ T cells from the spleens (Fig. 1A and C). Lung CD4+ T cells had a pattern of V usage similar to that of CD4+ T cells from the spleen (Fig. 1B and D). The TCR V usage of CD4+ T cells from the lungs was conserved, similar to the results found for lung CD8+ T cells, but the frequencies of some CD4+ TCR V subsets were more variable in the lungs than in the spleen (Fig. 1B and D). The total numbers of CD4+ and CD8+ T cells in the lungs were consistent for five uninfected mice, and so the absolute number of each V subset mirrored the frequencies (Fig. 1E and F). Thus, although the V TCR repertoire in lung T cells was similar to that in the splenic compartment and was conserved, there was some heterogeneity in the V TCR repertoire in lung T cells.
Lung TCR V repertoire during pulmonary C. neoformans infection. Our next objective was to determine whether particular V subsets were enriched in either the CD4+ or CD8+ T-cell populations during pulmonary C. neoformans infection. In response to pulmonary C. neoformans infection, resistant mice recruit both CD4+ and CD8+ T cells to the lungs (13). CD4+ T cells are relatively enriched, particularly late in the response (13). To control for differences in the number of T cells in the lungs of infected mice, we converted data to absolute numbers of each V subset per lung and determined the relative increase compared to uninfected controls for each subset. The absolute number and fold increase for CD4+ and CD8+ T cells expressing each of the V subsets were determined for mice at weeks 1, 3, and 5 postinfection. At week 1 postinfection, no V subsets were increased by more than onefold (data not shown), and there was no difference between subsets. At the peak of the T-cell response (week 3 postinfection), a modest increase in each of the CD8+ T-cell V subsets was observed, which represented 2.7- to 4.3-fold expansion compared to uninfected controls (Fig. 2D). Similarly, in CD4+ T cells, expansion of all V subsets occurred in response to infection (Fig. 2B). In contrast to the data for CD8+ T cells, the CD4+ V subsets were all expanded more than 11-fold compared to the CD4+ V subsets for uninfected control lungs (Fig. 2B). The number of V13+ CD4+ T cells was significantly expanded compared to the controls (>70-fold) (Fig. 2B); however, V13+ CD4+ T cells comprised a minority of the total CD4+ T-cell population in the lungs (6.3%) (Fig. 2A). This preferential expansion of V13+ CD4+ T cells was not observed at week 5 postinfection (Fig. 2F), and V13+ T cells were an even smaller proportion of the total lung CD4+ T cells (1.9%). Thus, during primary pulmonary C. neoformans infection, a diverse TCR V repertoire was maintained by both CD4+ and CD8+ T cells infiltrating the lungs.
Lung CD8+ TCR V repertoire during pulmonary C. neoformans infection in CD4+ T-cell-deficient mice. Pulmonary C. neoformans infection primarily affects individuals with compromised CD4+ T-cell immunity (5). To determine the effect of CD4+ T-cell deficiency on the resulting CD8+ TCR V repertoire, mice were made CD4+ T cell deficient by systemic administration of anti-CD4 depleting monoclonal antibodies as described in Materials and Methods. The expansion of CD8+ T cells belonging to the various TCR V subsets was assayed as described above. At week 3 postinfection, higher numbers of CD8+ T cells in each of the V subsets were present in the lungs compared to the numbers in CD4+ mice (Fig. 3A and 2D). The numbers represented a much greater expansion compared to uninfected controls (range, 6.9- to 42.0-fold), and V 2, 4, 10, and 14 all expanded more than 25-fold (Fig. 3A). At week 5 postinfection, V 2, 4, and 10 were expanded more than 35-fold (Fig. 3B). These numbers reflect total CD8+ T-cell expansion, which was exaggerated in the absence of CD4+ T cells. Thus, CD4+ T-cell deficiency resulted in expansion of all CD8+ V subsets compared to CD4+ mice, but the expansion was particularly pronounced for V subsets 2, 4, and 10.
TCR V repertoire during the secondary immune response to pulmonary C. neoformans infection. To determine whether mice maintained a diverse T-cell V repertoire during secondary infection, mice were infected with 103 CFU of C. neoformans and then housed in specific-pathogen-free conditions for 16 weeks to allow resolution of this primary infection. The mice were then given a secondary challenge consisting of 104 CFU, and the V repertoire was characterized 10 days after the secondary challenge. In the CD8+ T-cell compartment, very little expansion of CD8+ T cells was observed, and four of eight V subsets were contracted compared to uninfected controls (Fig. 4A). Whether increased numbers in the other four V subsets represented skewing of the CD8+ V repertoire in the secondary response is unclear, because the total number of CD8+ T cells at day 10 after the secondary challenge was similar to the number of cells in uninfected controls (data not shown). In the CD4+ T-cell compartment, preferential expansion of some V subsets was observed in individuals (Fig. 4B). For example, mouse 4 exhibited skewing of the CD4+ repertoire to V 4 and V 13, whereas mouse 3 preferentially expanded V 8.3 and V 14 (Fig. 4B). However, preferential usage of V subsets by CD4+ T cells was not consistent between individuals. Thus, during the secondary T-cell response to pulmonary C. neoformans infection, individuals exhibited skewing of the CD4+ T-cell repertoire, but consistent, preferential expansion of any V subset was not observed across the group.
Proliferative responses of T cells from immunized mice in response to C. neoformans antigens. The results described above demonstrated that a diverse TCR V repertoire was maintained in the T-cell response to pulmonary C. neoformans infection. Additionally, these results suggested that tracking any V subset would likely be a poor surrogate for antigen specificity in the T-cell response to pulmonary C. neoformans infection. Therefore, we shifted the focus of our investigation to determining whether we could identify a method for more specifically assaying the T-cell responses to C. neoformans.
To do this, we studied the proliferative responses of CD4+ and CD8+ T cells from mice immunized with C. neoformans. CBA/J mice were infected with 103 CFU of C. neoformans and allowed to recover from the infection. Previous studies demonstrated that there were enhanced T-cell responses to secondary C. neoformans infection in such mice (D. Lindell, M. Ballinger, R. McDonald, G. Towes, and G. Huffnagle, submitted for publication). At 16 weeks postinfection, single-cell suspensions were prepared from pooled lymph nodes and spleens of immunized mice. To assay for C. neoformans-specific responses, we generated a variety of antigen preparations from C. neoformans, as described in Materials and Methods. C. neoformans can persist in the lungs of resistant mice long after a primary infection (Lindell et al., submitted). The levels of cells recovered from the secondary lymphoid organs of mice immune to C. neoformans mice were below the detectable levels of viable yeast cells (<50 CFU in the pooled sample for five mice).
The proliferation of CD4+ and CD8+ T cells from mice at week 16 postinfection was assessed using a flow cytometry-based proliferation assay, as described in Materials and Methods. This assay has an advantage over [3H]thymidine incorporation assays because the proliferation of multiple cell populations can be determined independently in a single treatment well. Following 2 days in culture, there was minimal proliferation (as indicated by a decrease in CFSE staining) of either CD4+ or CD8+ T cells from immunized mice in the absence of stimulation (Fig. 5). In response to anti-CD3/anti-CD28 restimulation, a majority of CD4+ and CD8+ T cells proliferated (Fig. 5). A slight decrease in CFSE intensity was observed in both CD4+ and CD8+ T cells stimulated with C. neoformans lysate (Fig. 5). In contrast, after 4 days in culture, significant proportions of both CD4+ and CD8+ T cells from immunized mice had proliferated in response to C. neoformans lysate (Fig. 5).
Proliferative responses by both CD4+ and CD8+ T cells were also observed in response to a number of other C. neoformans-derived antigen preparations, including heat-killed organisms, culture filtrate antigen (CneF), and purified cryptococcal mannoprotein (Fig. 6A). These results demonstrated that a portion of CD4+ and CD8+ T cells in the secondary lymphoid tissues of immune mice proliferate in response to a number of C. neoformans antigen preparations.
Effector cytokine production by T cells from immunized mice in response to C. neoformans antigens. Our next objective was to determine whether the preparations of C. neoformans antigens (heat-killed organisms, lysate, CneF, or mannoprotein) could be used to assay effector cytokine production. To do this, lymphocyte suspensions from immune mice were cultured with uninfected adherent splenocytes in the presence of each stimulus, and the production of IFN- and TNF- was assayed by intracellular flow cytometry, as described in Materials and Methods. In response to each of the antigen preparations, CD4+ T cells produced both IFN- and TNF- (Fig. 6B). In contrast, CD8+ T cells produced only IFN- in response to C. neoformans antigens (Fig. 6C). These results demonstrate that T cells from the secondary lymphoid tissues of immune mice produced effector cytokines in response to nonviable C. neoformans antigen preparations.
Proliferation of CD4+ and CD8+ T cells from LALN of mice with pulmonary cryptococcal infections. Our next objective was to determine whether T cells from the LALN of mice during primary pulmonary infection proliferate in response to C. neoformans antigens. To do this, we assayed proliferation of LALN CD4+ and CD8+ T cells in response to C. neoformans lysate. The results obtained with immune mice suggested that maximal proliferation and effector cytokine production could be assayed using lysate, relative to the other antigen preparations (Fig. 6). Adherent lung cells from uninfected mice were cultured with LALN cells from mice 2 weeks after a primary infection, as described in Materials and Methods. Minimal proliferation was observed when LALN T cells were not stimulated; however, LALN T cells from infected mice proliferated in response to C. neoformans lysate (Fig. 7). We also investigated whether C. neoformans lysate induced proliferation from naive T cells. Although no such activity has been demonstrated for murine T cells, components of the C. neoformans cell wall have been shown to have mitogenic activity with human T cells (21, 25). After 4 days in culture, there was no difference in CFSE staining between unstimulated T cells from uninfected spleens and T cells stimulated with C. neoformans lysate (Fig. 7). T cells from uninfected spleens were capable of proliferation, however, and divided extensively in response to anti-CD3/anti-CD28 stimulation (Fig. 7). Both CD4+ and CD8+ T cells obtained from the LALN of mice at week 2 postinfection proliferated in response to C. neoformans lysate (Fig. 7). These results demonstrated that C. neoformans lysate did not induce mitogenic proliferation in naive murine T cells and that both CD4+ and CD8+ T cells recovered from the LALN proliferated in response to C. neoformans lysate. These data support the conclusion that a portion of the T cells in the LALN during pulmonary C. neoformans infection are antigen specific.
Effector cytokine production by CD4+ and CD8+ T cells from C. neoformans-infected mice. Our next objective was to determine the frequencies of CD4+ and CD8+ T cells producing the effector cytokines IFN- and TNF- in the lungs and secondary lymphoid tissues of C. neoformans-infected mice. To do this, T cells from infected lungs, LALN, spleens, uninfected lungs, and uninfected spleens were enriched by MACS or FACS, as described in Materials and Methods. T cells were cultured with adherent cells from uninfected control lungs in the presence of no stimulus or C. neoformans lysate. The results were expressed as the increase in the number of cytokine-positive cells compared with the number in unstimulated samples.
CD4+ T cells from infected lungs, but not CD4+ T cells from secondary lymphoid tissues, produced IFN- at a high frequency in response to CD3/CD28 restimulation (Fig. 8). CD4+ T cells in infected lungs produced TNF- in response to CD3/CD28 restimulation, but the frequency of TNF-+ CD4+ T cells in uninfected tissues was equal to or higher than the frequency of TNF-+ CD4+ T cells in infected lungs (Fig. 8). These results demonstrated that in response to CD3/CD28 restimulation, lung CD4+ T cells from infected mice produced IFN-. CD4+ T cells regulated the production of TNF- less stringently than they regulated the production of IFN-, however, as CD3/CD28 stimulation resulted in production of TNF- by CD4+ T cells from uninfected mice. CD8+ T cells from lungs and secondary lymphoid tissues produced IFN- in response to CD3/CD28 restimulation (Fig. 8). However, similar frequencies of CD8+ T cells from uninfected tissues produced IFN- in response to CD3/CD28 (Fig. 8). Thus, CD8+ T cells can produce TNF- and IFN- in response to low-dose CD3/CD28 restimulation in the absence of infection.
In response to C. neoformans lysate, CD4+ T cells from secondary lymphoid tissues did not produce IFN- or TNF-. Markedly higher frequencies of IFN- and TNF- production were observed with CD4+ T cells from infected lungs (Fig. 8). Although anti-CD3/anti-CD28 restimulation resulted in significant IFN- production by CD8+ T cells from the secondary lymphoid tissues (Fig. 8), CD8+ T cells from secondary lymphoid tissues restimulated with lysate were poor effector cytokine producers (Fig. 8). Similar to the results obtained with CD4+ T cells, only lung CD8+ T cells from infected lungs produced IFN- and TNF- in response to C. neoformans lysate (Fig. 8). These data show that C. neoformans lysate induces minimal effector cytokine production by T cells in uninfected mice. Furthermore, these results demonstrate that lung T cells from C. neoformans-infected mice produce effector cytokines in response to in vitro restimulation with C. neoformans lysate and that effector cytokine production occurs at a higher frequency than it occurs in T cells from secondary lymphoid tissues.
DISCUSSION
In this paper we report that in immunocompetent mice, a diverse TCR V repertoire is maintained throughout the primary response to pulmonary C. neoformans infection. While V 13 CD4+ T cells were preferentially expanded at week 3 postinfection, they comprised a minority of the overall lung CD4+ T-cell population. Furthermore, this preferential expansion did not persist until week 5 postinfection. CD4+ T-cell deficiency resulted in relative expansion of all CD8+ T-cell subsets. During the secondary immune response, preferential usage of CD4+ T-cell V subsets occurred in individuals, but not overall. Both CD4+ and CD8+ T cells from the secondary lymphoid tissues of immunized mice proliferated in response to a variety of C. neoformans antigens, including heat-killed whole C. neoformans, culture filtrate antigen (CneF), C. neoformans lysate, and purified cryptococcal mannoprotein. CD4+ and CD8+ T cells from the secondary lymphoid tissues of immune mice produced IFN- in response to these stimuli, and CD4+ T cells also produced TNF-. Both CD4+ and CD8+ T cells from the secondary lymphoid tissues of mice exhibiting a primary response to C. neoformans proliferated in response to C. neoformans lysate. In response to stimulation with C. neoformans lysate, lung CD4+ and CD8+ T cells produced the effector cytokines TNF- and IFN-.
Our studies demonstrated that a diverse TCR V repertoire is maintained throughout the primary response, but focusing of the TCR V repertoire occurred in CD4+ T-cell populations of individuals during the secondary immune response. Pulmonary infection of mice with H. capsulatum leads to significant skewing of the TCR V repertoire, as does protective vaccination with H. capsulatum heat shock protein 60 (9, 10, 23). Depletion of the preferentially expanded population in either case adversely affects clearance (9, 10, 23). Evidence obtained with other experimental systems, however, suggests that V preferences during primary infection are the exception rather than the rule. During primary Listeria monocytogenes infection, epitope-specific T cells have a diverse TCR V repertoire (4). In a secondary response, however, focusing of the V repertoire occurs in the responding antigen-specific T-cell populations (4). Similar to the results reported here, variability between genetically identical individuals was also observed (4). A T-cell response in which a common TCR V repertoire is found in all individuals has been termed a "public" repertoire, whereas a "private" repertoire is specific to an individual (6). In a number of recent studies workers have sought to determine the relative roles of private and public repertoires, chiefly in models of T-cell responses to viruses (14, 26). The CD8+ T-cell response to influenza in C57BL/6 mice is directed primarily against the immunodominant epitopes NP366 and PA224. The response to NP366 is characteristically public, whereas the response to PA224 is largely private (14, 15, 26). Although similar numbers of NP366- and PA224-specific T cells are generated in the primary response, the secondary response is dominated by the public NP366 response, suggesting that public specificity may play a more prominent role in secondary T-cell responses (2). However, private specificity may play a more important role in regulating differential responses to heterologous infections in genetically identical mice (15). Although in our study we did not utilize techniques such as spectratyping, the differences in V profiles between individual mice suggest that the CD4+ T-cell V repertoire during secondary C. neoformans infection could be characterized as largely private.
Our results indicated that during CD4+ T-cell deficiency, C. neoformans infection led to enhanced expansion of all CD8+ V subsets compared to the expansion observed with CD4+ T-cell sufficiency. Previous studies demonstrated that hyperexpansion of CD8+ T cells occurs in response to C. neoformans during CD4+ T-cell deficiency (18). The IFN--producing CD8+ T-cell effectors mediate a level of protection in a CD4+ T-cell-deficient host (18). Together, these results demonstrate that while the absence of CD4+ T cells increases the magnitude of the CD8+ T-cell response to C. neoformans infection, the CD8+ response to C. neoformans in a CD4+ T-cell-deficient host is also characterized by diversity.
If C. neoformans has superantigen activity in vivo in mice, we would have expected specific V subsets to be expanded in response to C. neoformans infection. Superantigens are microbially derived proteins which stimulate the proliferation of T cells in a non-antigen-specific manner via direct binding of the V region of the T-cell receptor (17). Superantigens may benefit a pathogen by inducing a high frequency of nonspecific T cells, which can interfere with the antigen-specific response (12). C. neoformans produces a mitogen (CnM) for human T cells, whose activity is localized to proteins found in the cell wall and membrane and can stimulate naive T-cell proliferation (20, 21). The mitogenic effect is similar to that of staphyloccocal enterotoxin B, a known superantigen, and requires phagocytosis and protein processing (24). In our study, we observed no proliferation by CD4+ or CD8+ T cells in response to C. neoformans lysate, even after 5 days in culture. Thus, our results suggest that C. neoformans mitogen is not active in mice.
Our data demonstrated that while T cells from the lymph nodes or spleens of mice at 2 weeks postinfection produced little TNF- and IFN- in response to stimulation with C. neoformans lysate, a significant number of T cells in the lungs produced each of these effector cytokines (Fig. 8). These results show that there is compartmentalization of T-cell effector function during pulmonary C. neoformans infection. T cells encounter antigen-presenting cells in the LALN bearing cognate antigen, where they respond by proliferating. Concomitant with trafficking to the lungs, both CD4+ and CD8+ T cells acquire an antigen-dependent effector cytokine-producing phenotype. Throughout the immune response to pulmonary C. neoformans infection, both CD4+ and CD8+ T cells maintain a diverse TCR V repertoire, and the response is driven by multiple C. neoformans antigens rather than by one or two immunodominant antigens.
ACKNOWLEDGMENTS
We thank Stuart Levitz and his laboratory for providing purified mannoprotein.
This work was supported by National Institutes of Health grants R01-HL065912 (to G.B.H.), R01-AI059201 (to G.B.H.), R01-HL051082 (to G.B.T.), and T32-AI07413 (to D.M.L.) and by a Department of Veterans Affairs merit grant (to G.B.T.).
FOOTNOTES
Corresponding author. Mailing address: Pulmonary and Critical Care Medicine, 6301 MSRB III, University of Michigan Medical Center, Ann Arbor, MI 48109-0642. Phone: (734) 936-9369. Fax: (734) 764-2655. E-mail: ghuff@umich.edu.
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