Abstract
Immunization with recombinant heat shock protein 60 (rHsp60) from Histoplasma capsulatum or a region of the protein designated fragment 3 (F3) confers protection from a subsequent challenge in mice. To determine the T cell repertoire involved in the response to Hsp60, T cell clones from C57BL/6 mice immunized with rHsp60 were generated and examined for Vβ usage by flow cytometry and RT-PCR. Vβ8.1/8.2+ T cells were preferentially expanded; other clones bore Vβ4, -6, or -11. When Vβ8.1/8.2+ cells were depleted in mice, Vβ4+ T cell clones were almost exclusively isolated. Measurement of cytokine production demonstrated that nine of 16 Vβ8.1/8.2+ clones were Th1, while only three of 13 non-Vβ8.1/8.2+ clones were Th1. In mice immunized with rHsp60, depletion of Vβ8.1/8.2+, but not Vβ6+ plus Vβ7+, T cells completely abolished the protective efficacy of Hsp60 to lethal and sublethal challenges. Examination of the TCR revealed that a subset of Vβ8.1/2+ clones that produced IFN-γ and were reactive to F3 shared a common CDR3 sequence, DGGQG. Transfer of these T cell clones into TCR α/β−/− or IFN-γ−/− mice significantly improved survival, while transfer of other Vβ8.1/8.2+ clones that were F3 reactive but were Th2 or clones that were not reactive to F3 but were Th1 did not confer protection. These data indicate that a distinct subset of Vβ8.1/8.2+ T cells is crucial for the generation of a protective response to rHsp60.
Histoplasma capsulatum (Hc)3 is a dimorphic, pathogenic fungus that causes disease in various mammalian species, including humans. Infection occurs via inhalation of conidial spores that transform into pathogenic yeasts within the mammalian host. Hc yeasts are readily phagocytosed by resident macrophages, where they survive and replicate unless the immune system is activated. The effective immune response to Hc relies on adaptive immunity and the collaboration between infected macrophages with T cells (1, 2). This interaction results in an increase in the amounts of Th1 cytokines, including IL-12, IFN-γ, and TNF-α, and the formation of granulomas that function to surround and contain the infection (3, 4, 5). In patients without underlying immune dysfunction, this response is usually successful at limiting the infection and preventing serious, disseminated disease. While the initial infection is contained, it does persist in the host in a dormant state within calcified granulomas and is capable of causing disease by reactivation if immunological dysfunction develops (2).
Protection against Hc infection can be achieved in mice by immunizing with Ag from a cell wall/cell membrane extract of cultured yeasts (6). Subsequent studies revealed that a particular Ag within the extract, heat shock protein 60 (Hsp60), is immunogenic and confers protection against infection (7, 8). Moreover, a region of Hsp60 between aa 172 and 443, termed fragment 3 (F3), is the protective domain of this protein (9). Studies have demonstrated Hsp60 to be a potent inducer of cellular immunity and an immunodominant Ag from Hc as well as other pathogenic organisms (10, 11, 12, 13). The protective response to Hsp60 and the resulting efficacy of vaccination are critically dependent on the presence of CD4+ T cells. It is believed that activated T cells produce IFN-γ which activates macrophages enabling the cells to kill intracellular yeasts.
While extremely diverse overall, T cells are organized as clonal populations that contain identical Ag specificity as determined by limited sequences, including 3 complementarity determining regions (CDR), of which CDR3, in the β-chain of the TCR, is believed to determine Ag-specificity (14, 15). Interaction with a defined epitope of an Ag by the TCR is required to activate the T cell to proliferate and generate cytokines, including IFN-γ. Previous work with Hc has indicated that biases in TCR repertoires are generated during the immune response to Hc. In a primary infection of Hc at the onset of CMI, Vβ4+ T cells are preferentially expanded in the lungs of mice (16). On the other hand, Vβ4+ and 6+ cells are important in secondary infection (17). The expansion of Vβ6+ T cells also occurs following immunization with the protective Ag F3, and they play an integral role in the protective response to this Ag (18).
In this study we analyzed T cells from recombinant Hsp60 (rHsp60)-immunized mice and demonstrated that T cells expressing Vβ8.1/8.2 are dominant. Among all T cells, regardless of their Vβ expression, there was wide variation in the ability to respond to F3, but Th1-producing clones were almost exclusively Vβ8.1/8.2+. Depletion of Vβ8.1/8.2+ T cells in mice abrogated the protective response to rHsp60 immunization in both lethal and sublethal models. In addition, transfer of a Vβ8.1/8.2 + clone, which was reactive to F3 and produced IFN-γ, to α/β T cell-deficient mice, restored protection from infection with Hc. These data indicate that a clonal subset of T cells that express the Vβ8.1/8.2 region of the TCR is sufficient to generate a protective response to rHsp60 of Hc.
Materials and Methods
Animals
Male C57BL/6 mice and athymic nude mice were purchased from the National Cancer Institute (Fredrick, MD). TCR α/β−/− and IFN-γ−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Athymic nude mice were used to produce ascites. Animals were housed under barrier conditions and were age- and sex-matched for all experiments. All animal experiments were performed in accordance with the Animal Welfare Act guidelines of the National Institutes of Health.
Hc culture
Hc strain G217B yeast cells were cultured and quantified as described previously (19). Following anesthesia with isoflurane, mice were infected intranasally (i.n.) with Hc. Infectious doses were 2 × 106 (sublethal) or 2 × 107 (lethal) yeasts in ∼35 μl of HBSS.
Recombinant Hsp60 and F3 production
Recombinant Hsp60 and F3 from Hc were generated as described previously (9). Briefly, genes expressing each protein were cloned in to the NdeI and BamHI sites of pET19b. Expression of recombinant protein was achieved by transforming Escherichia coli cells with the plasmid and growing the bacteria in Luria-Bertoni broth until an OD600 of 0.4–0.5 was reached. Isopropylthiogalactose was added to cultures at a final concentration of 1 mM and cells were grown for 3 h. Cells were harvested by centrifugation, and the resultant pellet was resuspended in a buffer containing 5 mM imidazole, 500 mM NaCl, and 20 mM Tris-Cl, pH 7.9. Samples were lysed by freeze-thaw cycles, followed by sonication. Soluble and insoluble fractions were separated by centrifugation at 20,000 × g.
The insoluble pellet was suspended and denatured in a buffer containing 6 M urea, 500 mM NaCl, 5 mM imidizole, and 20 mM Tris-Cl, pH 7.9. Denatured material was recovered in the supernatants following centrifugation at 20,000 × g and was subsequently filtered to remove particulate material. The protein was purified by metal-chelate chromatography using a Ni2+-Sepharose affinity column (His-Bind; Novagen, Madison, WI). The resulting protein was eluted with the same buffer used to suspend the insoluble pellet, but with 1 M imidizole. The eluate was dialyzed against buffer containing decreasing amounts of urea, the recombinant protein was concentrated by ultrafiltration, and the protein concentration was measured. Each protein contained <10 pg of LPS/μg of protein.
Splenocyte preparation
Splenocytes were obtained by teasing apart spleens between the frosted ends of two glass slides. Cells were washed three times with HBSS and resuspended in RPMI (BioWhittaker, Walkersville, MD) supplemented with 10% FBS, 1% l-glutamine, 1% sodium pyruvate, 1% nonessential amino acids, 5 × 10−5 M 2-ME, and 10 μg/ml gentamicin if used for the establishment or expansion of T cell lines and clones, but with only 10% FBS, gentamicin and l-glutamine if used in proliferation assays.
T cell line and clone production
Mice were immunized with 100 μg of rHsp60 s.c. and again 2 wk later. Spleen cells were harvested 2 wk after the final immunization and were stimulated with rHsp60 at 5 μg/ml for 2 wk in supplemented RPMI. Following the initial stimulation, fresh medium and Ag were added at 2-wk intervals with fresh irradiated spleen cells that served as APC. Individual T cell clones were obtained following several passages of the parent line by limiting dilution and were propagated as described above with the addition of 5% supernatant derived from rat splenocytes stimulated with Con A (Sigma-Aldrich, St. Louis, MO) for 48 h (20).
Proliferation assay
To each well of a microtiter plate, 2 × 104 resting T cell clones in 0.1 ml and 5 × 105 irradiated splenocytes in 0.1 ml as APC were added; 50 μl of either medium or Ag (F3 or rHsp60) was added to each well (final concentration of Ag in each well, 5 μg/ml). The cells were incubated for 72 h. Cells were pulsed 18 h before being harvested with the addition of 1 μCi [3H]thymidine (sp. act., 6.7 Ci/mmol; New England Nuclear, Boston, MA). Cells were harvested onto glass-fiber filter paper, and incorporation of radioactivity was measured by a liquid scintillation counter. Activity is presented as stimulation index: the cpm of cells stimulated with Ag divided by the cpm of cells in medium alone. Reactivity to F3 was defined as a stimulation index of a ≥2.5-fold increase in proliferation of cells compared with cells that were not stimulated with Ag.
Vβ analysis and TCR sequencing
Expression of the Vβ-chain on the surface of T cell clones was assessed by flow cytometry. Aliquots of each T cell clone containing ∼105 cells were incubated with anti-CD16 mAb (Fc block; BD PharMingen, San Diego, CA) and one of the biotinylated Abs against each of the Vβ families for 15 min at 4°C. After washing three times, the cells were incubated with streptavidin-PE for 15 min at 4°C and washed three times. Cells were then fixed with 1% paraformaldehyde. Fluorescence was measured using the FACSCalibur flow cytometer. Vβ expression was determined by examining the number of cells within the PE channel divided by the total number of cells counted.
Once the Vβ family was identified, RNA was isolated from the T cell clones using RNAzol (Biotecx, Houston, TX) lysis and chloroform extraction according to the manufacturer’s protocol. cDNA was synthesized by annealing RNA with 10 ng of an antisense primer specific to the Cβ region of the TCR and incubating with avian reverse transcriptase (Invitrogen, La Jolla, CA). The nucleotide sequence of each TCR cDNA was obtained using a sense primer specific to the expressed Vβ family and an antisense primer specific to the constant region of the β-chain as previously described (8). PCR products were purified and subcloned into pCR2.1-TOPO (Invitrogen). Several colonies were selected and used to isolate DNA. Multiple DNA samples from each T cell clone were submitted for sequence analysis by automated sequencing at University of Cincinnati DNA core facility.
Preparation of mAb
Production of mAb was achieved by injection of hybridoma cells into athymic nude mice. The hybridoma cell line KJ16 (Rat IgG1 anti-Vβ8.1/8.2) was provided by Dr. P. Marrack (National Jewish Hospital, Denver, CO), RR4-7 (Rat IgG2b anti-Vβ6) was a gift from Dr. O. Kanagawa (Washington University, St. Louis, MO), and TR3-10 (rat IgG2b anti-Vβ7) was provided by Dr. I. Weissman (Stanford University, Stanford, CA). mAb was purified using a protein G-Sepharose column (Amersham Pharmacia Biotech, Piscataway, NJ).
In vitro depletion of T cells
Mice were injected i.p. with 100 μg of mAb on days 7 and 3 before and on the day of immunization. Animals were subsequently injected once a week until the end of each experiment. Depletion of >95% of the T cell population was confirmed by flow cytometry.
Analysis of cytokine production
Quantitative organ culture
Mice were sacrificed and examined for fungal burden in lungs and spleens. Organs were harvested and homogenized in 5 ml HBSS. Organ homogenates were plated on blood-heart infusion agar plates at multiple 10-fold dilutions and incubated at 30°C until colony growth could be measured. Colony counts are given as mean log10 CFU per organ ± SE.
Adoptive transfer
T cell clones selected based on cytokine production and F3 reactivity were expanded to generate sufficient cells to inject 2 × 106 T cell clones i.v. into each TCR α/β−/− mouse. Mice were infected with 5 × 105 Hc yeasts i.n. 8 h later and observed for survival.
Statistical analysis
To analyze differences in fungal burden, a one-way ANOVA was performed. Under normalized conditions, the Tukey test was used to allow multiple comparisons among the different groups. Differences in survival were assessed by a log-rank test. Fisher’s exact test was used to compare the proportions of T cell clones.
Results
Vβ expression of Hsp60 T cell clones
To determine the TCR repertoire following rHsp60 immunization, T cells were harvested separately from two groups of mice, each immunized twice with 100 μg of rHsp60. T cells were propagated at 2-wk intervals, and individual T cell clones were isolated by limiting dilution. Analysis of Vβ expression of each T cell clone was determined by flow cytometry. Of the 29 clones isolated, the expression of Vβ8.1/8.2 was present in 56% of the T cell clones. Smaller proportions of the isolated T cell clones isolated expressed Vβ4, -6, -10, or -11 (Fig. 1⇓).
Vβ usage among T cell clones isolated following rHsp60 immunization. T cell clones were analyzed for Vβ expression using biotinylated isotype matched or mAb to Vβ8.1/8.2, followed by incubation with PE-labeled streptavidin. Bars represent the proportion of clones expressing a given Vβ.
The T cell clones were analyzed by RT-PCR to determine TCR sequence, including Jβ expression, and the sequence of the Ag recognition domain, CDR3 (Table I⇓). Several different Jβ families were represented, with no clear dominant family or correlation between Vβ and Jβ expression. Several CDR3 domains were also identified. In each case, clones sharing common CDR3 sequences shared the same Vβ and Jβ expression. The Jβ sequence among clones of the same Jβ family did manifest slight differences at the nucleotide level. Expression of a particular Vβ or Jβ region was not indicative of CDR3 sequence. The most conserved CDR3 sequences among Vβ8.1/8.2+ T cell clones were DGGQG (3 of 16) or GVGTP (5 of 16), while analysis of other Vβ families revealed no shared CDR3 sequences.
TCR sequence of T cell clones isolated following rHsp 60 immunization
In vitro characterization of T cell activity
To examine cytokine production by T cell clones, supernatants from clones were incubated with or without 5 μg/ml rHsp60, harvested after 48 h, and analyzed by ELISA (Fig. 2⇓). Among the 16 clones expressing Vβ8.1/8.2, nine were considered Th1, and seven were Th2. All of these clones produced measurable amounts of IFN-γ, and all but three produced IL-4. Among non-Vβ8.1/8.2+ clones, three of 13 produced no IFN-γ, while three others produced no IL-4. Unlike Vβ8.1/8.2+ clones, only three of 13 non-8.1/8.2 clones were Th1, while the remaining 11 were categorized as Th2.
Cytokine production by clones derived from rHsp60-immunized mice. Clones were stimulated with 5 μg/ml of rHsp60, and supernatants were harvested at 48 h. Supernatants were analyzed for cytokine production by ELISA and are presented as the mean picograms per milliliter ± SEM of stimulated-unstimulated cells. Vβ8.1/8.2+ clones are indicated by unshaded bars. Data represent results from four or more samples.
Production of GM-CSF was tightly linked to IFN-γ. In general, IFN-γ-producing clones also secreted GM-CSF. Similarly, production of IL-10 and IL-13 by T cell clones directly correlated with IL-4 production. All clones produced detectable levels of IL-13, and only one did not produce GM-CSF, while IL-10 was not produced by three of the T cell clones. None of the T cell clones stimulated with rHsp60 released more TNF-α than unstimulated cells.
In addition to cytokine analysis, each T cell clone was examined for F3 reactivity (Table I⇑). T cell clones were stimulated with F3 or rHsp60 to determine whether a correlation existed between TCR sequence and reactivity. Among the 16 Vβ8.1/8.2+ clones, nine were reactive to F3. Only two of 7 Vβ4+ clones reacted to F3, while one of two clones expressing Vβ6 or -10 demonstrated reactivity, and neither of the clones expressing Vβ11 responded. Using Fisher’s exact test, there was not a significant difference (p > 0.05) in F3 reactivity among T cell clones expressing different Vβ families. Reactivity to F3 correlated with clones expressing the CDR3 regions GVGTP and DGGQG.
T cell repertoire following Vβ8.1/8.2 depletion
To characterize the T cell repertoire in the absence of Vβ8.1/8.2+ T cells, rHsp60-immunized mice were administered with a Vβ8.1/8.2-specific mAb, KJ16. T cells were isolated from spleens 2 wk after the last immunization and cultured with rHsp60. Twenty-three clones were isolated, of which 75% (15 of 20) expressed Vβ4, and only 25% (5 of 23) expressed either Vβ6 or -7 (Table II⇓). Several of the Vβ4+ clones shared CDR3 homology, with six of the 15 clones expressing QEGTQ, three expressing DGQLG, and four expressing DRQGA. F3 reactivity was observed among only clones that expressed the QEGTQ sequence in the CDR3 region, but not other conserved CDR3 sequences.
T cell clones isolated from Vβ8.1/8.2-depleted mice
In addition to changes in CDR3 sequence and F3 reactivity, cytokine production by T cell clones isolated in the absence of Vβ8.1/8.2+ cells was also altered. The number of clones that produced IL-10, IL-4, or IL-13 was greatly reduced (Fig. 3⇓). Of the 23 clones, 14 produced no IL-4, 19 clones produced no IL-10, and 16 of the 23 generated no detectable IL-13. To ensure that altered levels of cytokine production were not due to problems of sensitivity using the ELISA, clones 20 (Th1), 12 (Th2), and 7 (Th2), which were isolated from Vβ8.1/8.2-intact mice, were run in parallel to clones isolated from depleted mice to directly compare levels of production. The results demonstrated that clones from Vβ8.1/8.2-intact mice had cytokine production equivalent to that observed in earlier experiments, but the amount of Th2 cytokines generated from depleted mice was consistently lower. IFN-γ production was detected in 11 of 23 clones, of which all were classified as Th1. GM-CSF was produced by 10 of 23 clones, of which eight were classified as Th1. Unlike the Th2 cytokines, the amount of GM-CSF or IFN-γ produced among these clones was similar to the amount generated by rHsp60 clones from Vβ8.1/8.2-sufficient mice.
Cytokine production by clones derived from Vβ8.1/8.2-depleted mice. Clones isolated from depleted mice were stimulated with 5 μg/ml of rHsp60, and supernatants were harvested 48 h later. Supernatants were analyzed for cytokine production by ELISA and are presented as the mean picograms per milliliter ± SEM of stimulated-unstimulated cells. Clones H7, H12, and H20 were derived from 8.1/8.2-intact mice and were run in parallel as an internal control. Data represent results from four or more samples.
Role of Vβ8.1/8.2 cells in Hc infection
To determine whether Vβ8.1/8.2+ cells were required for the generation of the protective immune response to rHsp60, immunized mice were injected with mAb to Vβ8.1/8.2+ cells and Vβ6+ and Vβ7+ cells or were given rat IgG. Two weeks after the second immunization, mice were challenged with a lethal inoculum of Hc (2 × 107) i.n. and observed for survival. All mice that had not been immunized with rHsp60 succumbed to infection by 11 days postinfection (dpi; Fig. 4⇓A). Similarly, immunized mice depleted of Vβ8.1/8.2+ T cells did not survive beyond 12 dpi. Conversely, all mice immunized with rHsp60 and injected with rat IgG or mAb to Vβ6 and 7+ T cells survived. At 40 dpi, lungs and spleens were harvested from the surviving animals and examined for fungal burden. In both groups the number of CFU was below the level of detection (102).
Effect of Vβ8.1/8.2 depletion on rHsp60-immunized mice. Mice (n = 6) were immunized; treated with rat IgG, mAb to Vβ8.1/8.2, or mAb to Vβ6 and -7; and infected i.n. with 2 × 107 Hc yeast cells (A), and survival was monitored for 40 days. B, Immunized and depleted mice were infected with 2 × 106 Hc yeasts in parallel with unimmunized controls. Lungs and spleens were harvested at 7 and 14 dpi and assayed for Hc CFU. The data represent the mean ± SEM. ∗, p < 0.05 compared with rHsp60-immunized mice given either rat IgG or mAb to Vβ6 and -7.
To determine whether elimination of Vβ8.1/8.2+ T cells was associated with increased fungal burden, rHsp60-immunized mice were administered rat IgG depleted of Vβ8.1/8.2+ or Vβ6 and Vβ7+ T cells. Two weeks following the second immunization with rHsp60, mice were infected with 2 × 106 Hc yeasts i.n. and sacrificed at 7 and 14 dpi. Fungal burden in lungs and spleens of mice depleted of Vβ8.1/8.2+ T cells was not significantly different (p > 0.05) from that in unimmunized controls on days 7 and 14 (Fig. 4⇑B). The lungs and spleens of mice immunized with rHsp60 and given rat IgG or mAb to Vβ6 and Vβ7 contained significantly fewer CFU (p < 0.05) than unimmunized controls or those given mAb to Vβ8.1/8.2. No difference in fungal recovery was detected between rHsp60-immunized mice and immunized mice that lacked Vβ6 and Vβ7 (p > 0.05) on both days 7 and 14.
Depletion of the entire Vβ8.1/8.2+ repertoire could have a profound effect on the ability to mount an effective immune response. To ensure that the increase in fungal burden and loss of survival was attributable to the specific response to Hsp60 and not a general defect in the immune system, unimmunized mice were depleted of Vβ8.1/8.2+ cells. These mice in parallel with nondepleted controls were infected with a sublethal inoculum, and the fungal burden was determined at 7 dpi. No significant (p > 0.05) difference was observed between depleted and intact animals in either lungs or spleens. The mean log10 CFU (±SEM) in lungs (6.1 ± 0.21) and spleens (4.7 ± 0.10) from Vβ8.1/8.2-depleted mice did not differ (p > 0.05) from that in lungs (6.0 ± 0.12) and spleens (4.6 ± 0.15) of infected controls.
Adoptive transfer of Vβ8.1/8.2+ T cells
To determine whether individual subsets of Vβ8.1/8.2+ T cells were capable of conferring a protective response, individual clones were injected into TCR α/β−/− mice that were infected with 5 × 105 Hc. T cell clones were selected based on F3 reactivity and cytokine production. In control mice that were not injected with T cells, all mice succumbed to infection by day 32 (Fig. 5⇓A). Mice that were injected with clone 17 that produced IFN-γ but was not reactive to F3 did not survive beyond 30 days (p > 0.05 compared with controls). Transfer of clone S cells that were reactive to F3 but produced IL-4 caused an acceleration of infection, and no mouse survived beyond 22 days (p < 0.05 compared with controls). Mice that were injected with clone D, which was reactive to F3 and produced IFN-γ, prolonged survival (p < 0.05) beyond that of control animals, with mean survival reaching 46 days.
Adoptive transfer of T cell clones into TCR αβ−/− (A) and IFN-γ−/− (B) mice. T cell clones S (Th2, F3+), D (Th1, F3+), or 17 (Th1, F3−) were injected i.v. into TCR α/β−/− (n = 6) or IFN-γ−/− mice (n = 5). Following transfer of cells, mice were infected i.n. with 5 × 105 Hc yeast cells and monitored for survival.
In an alternative model, Vβ8.1/8.2+ T cell clones were injected into IFN-γ−/− mice to determine whether protection could be restored in animals highly sensitive to Hc infection (5). Clones S, 17, and D were transferred. All mice receiving no T cells succumbed to infection with 5 × 105 Hc by day 11 (Fig. 5⇑B). IFN-γ−/− mice that received clone S or clone 17 were similar to mice that received no cells. Transfer of clone D dramatically enhanced survival (p < 0.05). All animals survived beyond 35 dpi, and only 40% succumbed by 40 dpi, at which time the study was terminated. The lungs and spleens of the surviving mice were analyzed for the presence of Hc, and in two of the three mice they contained <102 CFU. The remaining animal had ∼106 CFU in lungs and spleen.
Discussion
Both primary and secondary immune responses to Hc are dependent on T cell activation and production of cytokines that activate phagocytes to express antifungal activity. The ability of a T cell to respond to an Ag relies on the recognition of specific epitopes presented by APC, and this interaction causes activation and expansion of Ag-specific T cell clonotypes. Native or rHsp60 is a protective Ag that elicits a T cell response and is associated with increased production of IFN-γ, IL-10, and IL-12 (7, 8, 21). In the results presented here, rHsp60 from Hc caused the expansion of murine T cells that predominantly express the Vβ8.1/8.2 region of the TCR. Elimination of Vβ8.1/8.2+ cells abolished the protective efficacy following immunization with rHsp60 and led to increased fungal burden in the lungs and spleens of mice. More important, subsets of Vβ8.1/2+ T cells that were F3 reactive and produced IFN-γ, specifically those expressing the DGGQG CDR3 region, were able to confer a protective response to T cell- or IFN-γ-deficient mice. Thus, defined populations of T cells are involved in the generation of the protective response to rHsp60.
The importance of clonal T cell expansion in response to a pathogen (22, 23, 24, 25, 26) or as a cause of an autoimmune condition (27, 28) has been documented in several experimental models. Vβ8.1/8.2+ and Vβ10+ T cells are expanded during primary and memory response to lymphocytic choriomeningitis virus (29). A specific T cell repertoire also has been identified following infection with Leishmania major and has been correlated to a bias in Th2 production in susceptible mice (30, 31). However, the functional properties of the expanded repertoire in a protective immune response have not been rigorously addressed. We have reported that Vβ4+ T cells are preferentially expanded in the lungs of mice during a primary infection with Hc and that depletion of these cells causes an increase in fungal burden during primary infection (16). In secondary infection, Vβ4+ and Vβ6+ cells are important in protection (17). In addition to infection models with Hc, immunization with the protective fragment F3 drives the expansion of a specific Vβ repertoire. Following immunization with F3, only Vβ6+ and Vβ14+ T cells are isolated from infected mice. Depletion of Vβ6+ cells causes a complete abrogation in the protective response of F3 immunization, indicating that these cells are functionally required for the protective response to F3 (18).
Immunization with rHsp60 produced a very different profile in the TCR repertoire compared with immunization with F3 in the same strain of mouse. The response to rHsp60 was dominated by Vβ8.1/2+ T cells, whereas Vβ6+ cells were the preponderant TCR found following F3 immunization. The contributions of these two dominant Vβ families to the protective responses induced by each of their specific Ag were similar. In both, elimination of the Vβ population led to the abolition of protection. The absence of Vβ6+ cells was associated with an inability to isolate reactive T cell clones, indicating that this family was necessary for clonal expansion. A similar finding was observed in DBA/2 mice immunized with peptide 110–121 from sperm whale myoglobin. Elimination of the dominant Vβ8.2+ family blunts Ag responsiveness in vitro (32). Conversely, depletion of Vβ8.1/8.2+ cells from mice immunized with rHsp60 did not prevent the emergence of multiple Ag-reactive clones. Of the clones isolated from Vβ8.1/8.2+-deficient mice, Vβ4+ cells emerged with the highest frequency. These clones, however, showed a more limited diversity in terms of CDR3 sequences and Vβ expression. Despite the ability to generate rHsp60-reactive clones from Vβ8.1/8.2+-depleted mice, no other Vβ family was able to compensate for the loss of these cells in vivo.
Despite the multiple differences among T cell clones isolated, there were common CDR3 sequences found between F3- and rHsp60-isolated clones. After F3 immunization, 83% of the CDR3 sequences contained a GG region, and among F3-reactive clones isolated following immunization with rHsp60, 28% had a GG sequence in the CDR3 region. However, while the F3 clones were Vβ6+, Hsp60 clones that contained a GG region of the CDR3 expressed Vβ8.1/8.2 or -10. The difference in proportion of GG-containing clones could be an example of differences in the abundance of epitopes presented following immunization with the intact protein.
Vβ4+ T cell clones were consistently isolated from mice immunized with rHsp60 in the presence or the absence of Vβ8.1/8.2+ cells. Vβ4+ cells are preferentially expanded in the lungs of mice during primary infection, suggesting that this family of T cells recognizes an important Ag (8). Despite the importance of Vβ8.1/8.2+ cells following rHsp60 immunization, these cells were not significantly expanded during primary infection. While Vβ4+ clones were frequently isolated in these experiments, the results suggest that these cells, like Vβ6+ cells, cannot compensate for the loss of Vβ8.1/8.2+ cells and are not required for the protective response to rHsp60. Additional experiments have suggested that the Vβ4+ populations obtained from spleens in these experiments differ from those expanded in the lung during primary infection, and that the lung-derived cells recognize an Ag other than Hsp60, which may also be crucial in generation of the primary immune response. This result is in agreement with additional data that most Vβ4+ T cells isolated from lungs of infected mice show little or no reactivity to Hsp60 (M. Scheckelhoff, unpublished observation). Studies are currently underway to determine the Ag specificity and CDR3 sequences of lung-derived Vβ4+ T cells.
The protective response to rHsp60 requires the production of type 1 cytokines, especially IFN-γ (5, 21). Only T cells expressing Vβ8.1/8.2+ demonstrated higher proportions of IFN-γ production among the clones. Only three of the 13 non-Vβ8.1/8.2+ T cell clones generated were classified as Th1. In addition, while other Vβ families demonstrated IFN-γ production, these collective T cell populations were not able to compensate for the loss of Vβ8.1/8.2+ cells. This finding does not exclude the possibility that individual Th1 clones that do not express Vβ8.1/8.2 may be protective. Among Vβ8.1/8.2+ clones, the production of IFN-γ alone was not indicative of protection. Transfer of T cell clones demonstrated that Th1+ that were also reactive to F3 prolonged survival, while Th1+ clones that did not respond to F3 did not extend survival beyond that of infected control animals. These data suggest that following interaction with Ag, Th1+ Vβ8.1/8.2+ cells are activated and probably function to activate phagocytes to stimulate antifungal activity, but only those that produce Th1 cytokines and are reactive to F3 are involved in the protective response to rHsp60. F3 reactivity, however, is not alone indicative of protective immunity, as transfer of an F3-reactive 8.1/8.2+ clone that produced IL-4 and IL-10 into TCR α/β−/− mice caused the animals to succumb to infection at a faster rate than control mice that received no transferred cells. These results support the finding that excess production of IL-4 and/or IL-10 can be deleterious to control of Hc infection (4, 21). However, transfer of these clones does not accelerate the loss of survival in IFN-γ−/− mice. This finding is most likely the result of the Th2-biased environment already present in animals lacking IFN-γ. Hence, specific clonotypes of Vβ8.1/8.2+ T cells are capable of generating a protective response in a highly organized manner that is controlled through cytokine production among these and other Hsp60-reactive clones.
Among Vβ8.1/8.2+ clones, 15 of 16 demonstrated dual production of IL-10 and IFN-γ. Recent work has demonstrated that T cells that produce both these cytokines may play an important regulatory role in the generation of a protective immune response. Following infection with L. major or Mycobacterium tuberculosis, IL-10 has been suggested to play a role in controlling the Th1 response to prevent immunopathology in host tissues, but may also prevent sterilization of the tissue and allow dormancy and persistence (33, 34). Evidence that these cells were pivotal in the immune response was demonstrated by the lack of clones that produce both IL-10 and IFN-γ isolated when Vβ8.1/8.2+ cells were depleted before immunization. All but five of the clones derived from depleted mice produced IFN-γ, and 13 of 22 were classified as Th1, suggesting that compensation for Th1 production may be possible. However, only two of the 22 clones isolated from depleted animals produced both IL-10 and IFN-γ. This assertion is supported by the finding that neutralization of IL-10 interferes with the efficacy of rHsp60 immunization (21).
The results generated here demonstrate that vaccination with the protective protein Hsp60 in C57BL/6 mice can be directly correlated to the presence or the absence of Vβ8.1/8.2+ T cells expressing a specific CDR3 region (in this case, DGGQG). This finding has important implications for vaccine development for Hsp60 as well as other protein Ags that require T cell activity. While Vβ usage may vary depending on genetic differences among populations, the CDR3 region that determines epitope specificity will be shared among these cells, and therefore serve as an indicator for a protective response. In this example, the Vβ family identified is useful in terms of identification and categorization, but the efficacy remains dependent on the CDR3. Correlation of a vaccine and a specific, protective repertoire found within the population can be used as an indicator for an effective vaccine and reduce the requirement for extensive efficacy trials. Conversely, proteins that activate TCR repertoires that are found only in restricted populations will probably have limited success. In addition, the experiments presented here indicate that an epitope-specific subset of T cells can successfully direct the protective response to a single immunogenic Ag, suggesting that under favorable circumstances, the development of complex multivariant vaccines may not be necessary.
In summary, a repertoire of T cells expressing Vβ8.1/8.2 are generated following rHsp60 immunization and are required for the efficacy of rHsp60-induced protection. Among these cells, a defined subset that produces IFN-γ and is F3 reactive has been shown to prolong survival in mice and is likely to provide the functional attributes required for protective immunity.
Footnotes
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↵1 This work was supported by Grants AI34361 and AI42747 from the National Institutes of Health and by a Merit Review from the Department of Veterans Affairs.
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↵2 Address correspondence and reprint requests to Dr. George S. Deepe, Jr., Division of Infectious Diseases, University of Cincinnati College of Medicine, Cincinnati, OH 45267. E-mail address: george.deepe{at}uc.edu
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↵3 Abbreviations used in this paper: Hc, Histoplasma capsulatum; CDR3, complementarity-determining region; dpi, days postinfection; F3, fragment 3; i.n., intranasal; rHsp60, recombinant heat shock protein 60.
- Received May 31, 2002.
- Accepted September 12, 2002.
- Copyright © 2002 by The American Association of Immunologists
References
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