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V6⁺ T Cells Are Obligatory for Vaccine-Induced Immunity to Histoplasma capsulatum¹

Division of Infectious Diseases, University of Cincinnati College of Medicine, and Veterans Affairs Hospital, Cincinnati, OH 45267

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We examined TCR usage to a protective fragment of heat shock protein 60 from the fungus, Histoplasma capsulatum. Nearly 90% of T cell clones from C57BL/6 mice vaccinated with this protein were V6⁺; the remainder were V14⁺. Amino acid motifs of the CDR3 region from V6⁺ cells were predominantly IxGGG, IGG, or SxxGG, whereas it was uniformly SFSGG for V14⁺ clones. Short term T cell lines from V6⁺-depleted mice failed to recognize Ag, and no T cell clones could be generated. To determine whether V6⁺ cells were functionally important, we eliminated them during vaccination. Depletion of V6⁺ cells abrogated protection in vivo and upon adoptive transfer of cells into TCR ^-/- mice. Transfer of a V6⁺, but not a V14⁺, clone into TCR ^-/- mice prolonged survival. Cytokine generation by Ag-stimulated splenocytes from immunized mice depleted of V6⁺ cells was similar to that of controls. The efficacy of the V6⁺ clone was associated with elevated production of IFN-, TNF-, and GM-CSF compared with that of the V14⁺ clone. More V6⁺ cells were present in lungs and spleens of TCR ^-/- on day 3 postinfection compared with V14⁺ cells. Thus, a single V family was essential for vaccine-induced immunity. Moreover, the mechanism by which V6⁺ contributed to protective immunity differed between unfractionated splenocytes and T cell clones.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Infection with the pathogenic, soil-based fungus, Histoplasma capsulatum (Hc),³ produces a wide spectrum of disease, ranging from a mild, influenza-like illness to a progressive, disseminated form that can be life-threatening if untreated. The extent of disease induced by Hc can be correlated to the number of fungal particles to which one is exposed. Prevention of infection from Hc requires sequestration of a contaminated area, application of toxic chemicals, and the use of respirators by workers (1). Another means of prevention is vaccination for high risk groups. In this regard we have reported that immunization of mice with the immunodominant Ag, heat shock protein (hsp)60, from Hc confers protection against a lethal challenge given either i.v. or by intranasal (i.n.) instillation (2). The protective effect of Hc hsp60 is contained within a domain, termed F3, that spans aa 172–443 (3).

T cells exert an extremely influential role in combating infection with this fungus. In experimental primary infection, CD4⁺ cells are necessary for survival; CD8⁺ cells contribute to optimal clearance (4, 5, 6, 7). In the lungs of mice infected with Hc, V4⁺ cells are significantly elevated during the period of infection when cell-mediated immunity is activated, and depletion of this population blunts the protective immune response (8). In secondary Hc, protective immunity is abrogated only when both CD4⁺ and CD8⁺ cells are eliminated. However, the protective immune response is impaired when V6⁺ or V6⁺ plus V4⁺ cells are depleted from the host (9).

Virtually nothing is known concerning the TCR usage to a protective Ag from this fungus or other fungi. To gain a better understanding of the cellular mechanisms associated with vaccine-induced immunity, we examined the TCR repertoire of cells that react with F3. In this way we can begin to understand the underlying mechanism(s) by which this fragment expresses protection. The results demonstrate that a very high proportion of F3-reactive monoclonal cells from immunized C57BL/6 mice are V6⁺. These cells are crucial both to the generation of Ag-reactive cells in vitro and to the protective efficacy of F3.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Five- to 6-wk-old C57BL/6 and TCR ^-/- mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were housed in isolator cages and were maintained by the University of Cincinnati Department of Laboratory Animal Medicine, which is accredited by the American Association for Accreditation of Laboratory Animal Medicine. All animal experiments were performed in accordance with the Animal Welfare Act guidelines of the National Institutes of Health.

Preparation of Hc and infection of mice

Hc yeasts (strain G217B) were prepared as previously described (7). This strain is a prototypical virulent strain of this fungus (7). To produce a sublethal infection in naive mice, animals were infected i.n. with 2.0 x 10⁶ Hc yeasts in a 30-µl volume. A lethal inoculum of 1.25 x 10⁷ yeasts was used in some experiments with immunocompetent C57BL/6 mice.

Preparation of F3

Cloning and expression of F3 have been described previously (3). Briefly, the gene fragment was cloned into the NdeI and BamHI sites of pET19b. To express recombinant protein, Escherichia coli harboring the plasmid was grown at 37°C in Luria-Bertoni broth until an OD₆₀₀ of 0.4–0.5 was achieved. Subsequently, isopropyl-thiogalactose was added to cultures at a final concentration of 1 mM. Cultures were continued for 3 h. Cells were harvested by centrifugation at 5000 x g. The pellet was suspended in a buffer consisting of 5 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl, pH 7.9, and was lysed by freeze-thaw cycles followed by sonification. Soluble and insoluble fractions were separated by centrifugation at 20,000 x g.

The insoluble pellet was suspended in a buffer consisting of 6 M urea, 500 mM NaCl, 5 mM imidazole, and 20 mM Tris-HCl, pH 7.9. The denatured material was recovered in supernatants after centrifugation at 20,000 x g and then filtered to remove particulate material. The protein was purified by metal chelate chromatography using Ni²⁺-Sepharose affinity column (His-Bind; Novagen, Madison, WI). The recombinant protein was eluted with the same buffer as described above, except that it contained 1 M imidazole. The eluate was dialyzed against buffer containing decreasing amounts of urea. The eluate was concentrated by ultrafiltration, and protein concentration was determined. F3 contained <10 pg LPS/µg protein.

Organ culture for Hc

Recovery of Hc was performed as previously described (7). Fungal burden was expressed as mean CFU per whole organ ± SEM. The limit of detection is 10² CFU.

Preparation of mAb

Rat anti-mouse V6 chain (rat IgG2b, clone RR4-7) was provided by Dr. Osami Kanagawa (Washington University, St. Louis, MO). Rat anti-mouse V7 (rat IgG2b, clone TR 310) was provided by Dr. Irving Weissman (Stanford University, Stanford, CA). Ascites was prepared in nude mice. The IgG fraction was purified using a protein G-agarose column (Amersham-Pharmacia, Piscataway, NJ). The concentration of mAb was determined by ELISA using rat IgG as standard.

Immunization

Mice were immunized s.c. with F3 or, as a control, BSA. Both Ag were suspended in adjuvant containing monophosphoryl lipid A, synthetic trehalose dicorynomycolate, and cell wall skeleton (Ribi Immunochem, Hamilton, MT) at a concentration of 1 mg/ml. Animals were injected s.c. with 0.1 ml emulsion (100 µg protein) twice. Injections were separated by 2 wk, and mice were infected 2 wk after the last immunization.

Splenocyte preparation

Spleen cells were isolated by teasing apart spleens between the frosted ends of two ground glass slides. Cells were washed three times in HBSS and resuspended in RPMI 1640 containing 10% FBS, 1% nonessential amino acids, 1% sodium pyruvate, 5 x 10^-5 M 2-ME, and 10 µg/ml gentamicin (complete medium) if the cells were to be used for the establishment of T cell lines. For adoptive transfer experiments, cells were suspended in HBSS.

Isolation of lung leukocytes

Lungs were excised after flushing circulating leukocytes by injecting of 3 ml HBSS into the right ventricle. Lungs were minced apart in 10 ml of RPMI, and a single-cell suspension was obtained by forcing the lung fragments through needles of progressively smaller gauge, followed by filtration through a 60-µm nylon mesh. Leukocytes were purified by a 600 x g centrifugation through a discontinuous 40/70% Percoll gradient and were enumerated with a hemocytometer.

Establishment of T cell lines and clones

T cell lines and clones were initiated and maintained as previously described (10). T cells were propagated in the presence of irradiated splenocytes (2.0 x 10⁶), 20 µg/ml F3 or 125 µg/ml OVA (for the OVA-reactive clone SOG), and 5% IL-2 enriched supernatant. The source of IL-2 was produced by stimulating Lewis rat splenocytes with 5 µg/ml Con A for 48 h. Supernatants were harvested and 20 mg/ml -methyl-mannoside/ml was added. This preparation was filter-sterilized and stored at -70°C.

Proliferation assays

Resting T cells were suspended in RPMI 1640 supplemented with 10% FBS and 10 µg/ml gentamicin. To each well of a microtiter plate were added 2 x 10⁴ T cells in 0.1 ml, 5 x 10⁵ irradiated splenocytes in 0.1 ml, and 50 µl F3 (the final concentration in a well was 20 µg/ml). Cells were incubated for 72 h; 16 h before cell harvest, 1 µCi [³H]thymidine (sp. act., 6.7 Ci/mmol; New England Nuclear, Boston, MA) was added to each culture. Cells were collected on glass-fiber filters with a semiautomated harvester, and uptake of radioactivity was measured by a liquid scintillation counter. The stimulation index equals the cpm of cells in response to Ag minus the cpm of cells in medium alone.

RNA extraction

Cells (1 x 10⁶) were incubated in 0.2 ml RNAzol (Biotecx Laboratories, Houston, TX). RNA was extracted with chloroform and precipitated following the manufacturer’s protocol. RNA was resuspended in nuclease-free water, and the nucleic acid yield and purity were determined by OD₂₆₀ and the OD₂₆₀/OD₂₈₀ ratio. Samples were kept at -70°C until processed.

RT-PCR of V families

One microgram of total RNA was annealed with 10 ng of an antisense primer complementary to the constant region of the -chain (C1) of TCR. First-strand cDNA synthesis was performed with avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) and dNTPs. Aliquots of 1 µl of the RT reaction were used as template in 20 parallel PCR. Each tube contained a common nested antisense primer specific to the constant region of the -chain (C2) and each of 20 V-specific sense primers (9), dNTPs, and Taq polymerase (Life Technologies, Gaithersburg, MD). Reactions were denatured at 94°C for 45 s, annealed at 60°C for 45 s, and extended at 72°C for 60 s. The number of cycles necessary to produce a visible signal without saturation was determined in preliminary experiments. Between 28 and 32 cycles were used for most samples. The primers and their sequences have been previously published (11).

The presence of a V-specific PCR product was determined by Southern blot: 5 µl of each PCR was electrophoresed in 1% agarose gels, blotted onto nylon membranes (Roche, Indianapolis, IN), and hybridized with a digoxigenin-labeled DNA probe specific to the C region of the TCR. After washing in 0.1% SSC at 65°C, the signal was revealed with alkaline phosphatase-conjugated anti-digoxigenin Fab (GENIUS system; Roche) and the chemiluminescence substrate CDP Star (Roche). Light production was measured directly with a ChemiImager 4000 instrument (Alpha Innotech, San Leandro, CA).

Sequencing the TCR

The PCR product from V6⁺ or V14⁺ cells was cloned into pCR2.1 TOPO TA and transformed in TOP10F' cells (Invitrogen, Carlsbad, CA). For each T cell clone, plasmids from at least three colonies were sequenced.

FACS analysis

Cells were adjusted to 5 x 10⁵/200 µl in PBS containing 2% BSA and 0.02% sodium azide and stained with 0.5 µg of one of the following FITC-labeled mAbs (PharMingen, San Diego, CA): anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), or biotin-conjugated anti-V6 (clone RR4-7) or anti-V14 (clone 14-2), followed by incubation with streptavidin-PE . To determine the percentage of cells expressing V6 or V14 following adoptive transfer into TCR ^-/- mice, lung leukocytes and splenocytes were adjusted to 5 x 10⁵/200 µl in PBS containing 2% BSA and 0.02% sodium azide and stained with allophycocyanin-conjugated anti-CD3 (clone 145-2C11) or with biotin-conjugated anti-V6 or anti-V14, followed by incubation with streptavidin-PE. Staining with isotype-matched rat IgG or IgM mAb was performed in parallel. The samples were washed and fixed in 2% paraformaldehyde until analyzed on a flow cytometer.

In vivo depletion

Groups of mice were depleted of V6⁺ or V7⁺ T cells by injection of 150 µg RR4-7 or an equal amount of TR-310 mAb i.p. on days -7 and -3 and on the day of immunization. Mice were injected with mAb once a week thereafter until the end of each experiment. Control mice were given an equal amount of rat IgG i.p. The efficiency of depletion was >95% as determined by flow cytometry.

Adoptive transfer

Splenocytes were suspended in HBSS and injected i.v. into mice 8 h before i.n. infection. With splenocytes, 2 x 10⁷ cells/recipient were injected; for T cell clones, 2 x 10⁶/recipient were injected.

Generation of cytokine-containing supernatants

Splenocytes from F3-immunized mice treated with mAb to V6 or with rat IgG were resuspended in complete medium at a concentration of 3 x 10⁶/ml. One milliliter of cells was added to each well of a 24-well plate, and cells were incubated in the presence or absence of F3 (20 µg/ml). To generate supernatants from T cell clones, 10⁵ T cells were incubated with 2.0 x 10⁶ irradiated splenocytes in the presence or the absence of 20 µg/ml F3. One milliliter of cells was dispensed into each well of 24-well plate. All cultures were incubated for 48 h at 37°C in 5% CO₂. Supernatants were harvested, filter-sterilized, and stored at -70°C until assayed.

Cytokine analysis

Commercially available ELISA kits were used to measure IFN-, IL-4, TNF-, GM-CSF (Endogen, Cambridge, MA), and IL-10 (PharMingen, San Diego, CA). The data for cytokine measurement were expressed as the cytokine by subtracting the amount of cytokine detected in medium alone from that found in supernatants of Ag-stimulated cells.

Statistics

Student’s t test was used to compare groups if the data achieved normality; otherwise, the Wilcoxon rank-sum test was used. Survival data were analyzed using the log-rank test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TCR usage of F3-reactive clones

Groups of mice were immunized with F3, and T cell lines were established by propagation in fresh medium, Ag, and a source of IL-2 every 2 wk. After 6 passages the lines were cloned at 1 and 0.3 cells/well. All positive wells were expanded and assessed for reactivity. Eighteen clones that reacted with F3 from 2 independent lines were isolated and analyzed for V expression by RT-PCR and Southern blot. Among the 18 clones, 15 expressed V6 and three expressed V14 (Table I). V expression was confirmed by FACS analysis. All clones were CD4⁺.

CDR3 and J sequence of the F3-reactive clones

The sequences of the CDR3 and J regions were determined by PCR amplification of cDNA using a V6 sense primer combined with a C antisense primer. The resulting bands were gel-purified, cloned, and transformed into E. coli. Several colonies from each T cell clone were sequenced in both directions. Analysis of the clones indicated that six of 15 V6⁺ T cell clones expressed J2.3, four expressed J2.2, three expressed J2.6, and two expressed J1.3. All three of the V14⁺ clones expressed J2.4 (Table I). The amino acid composition of the CDR3 region from V6⁺ clones revealed the motif of IxGGG or IGG. The motif of SFSGG was found for each of the V14⁺ clones (Table I).

TCR usage and elimination of V6⁺ cells in F3-immunized mice

We sought to determine whether elimination of V6⁺ cells from F3-immunized mice would alter V expression by F3-reactive clones. Mice were depleted of V6⁺ cells by treatment with mAb to it and were immunized with F3. In six independent attempts we were unable to establish T cell lines that could be propagated longer than 1 month. Moreover, in proliferation assays the short-term lines from mice lacking V6⁺ cells failed to proliferate in response to F3. The stimulation index never exceeded 1 at each concentration of Ag tested (1–250 µg/ml). As an example, the mean response (±SEM) by splenocytes from F3-immunized mice that lacked V6⁺ cells in the presence of 20 µg/ml F3 was 12,028 ± 1,129 cpm, and in the absence of Ag the response was 12,778 ± 766 cpm. In contrast, the mean response by one of the F3-reactive lines to F3 was 16,486 ± 409 cpm, whereas the response by cells in medium alone was 2,618 ± 906 cpm.

To determine whether elimination of any V family would alter the ability to establish F3-reactive clones or affect the TCR usage, we depleted mice of V7⁺ cells and immunized with F3. T cell clones were isolated from V7⁺ cell-depleted mice. Ten F3-reactive clones were analyzed for V expression. All 10 clones were V6⁺, and nine expressed the J 2.2 sequence, whereas one was J1.2. The amino acid sequence of the CDR3 was SHAGG or SPSGG for the J2.2⁺ clones and RDN for the J 1.2⁺ clone (Table II).

View this table: [in this window] [in a new window]   Table II. Analysis of F3-reactive clones from mice depleted of V7 cells

In vivo effect of V6⁺ cells in F3-immunized animals

Because V6⁺ cells were central in establishing T cell reactivity to F3, we endeavored to determine whether their presence was essential for host protection following F3 immunization. Mice were treated with mAb to V6 or rat IgG and injected s.c. with F3. Two weeks after immunization, mice were challenged with 2 x 10⁶ Hc yeasts. One week later, mice were sacrificed, and the fungal burden in lungs and spleens was determined. These organs from mice immunized with F3 contained significantly fewer (p < 0.01) CFU than controls. Depletion of V6⁺ cells abrogated the protective efficacy of F3 in both lungs and spleens (Fig. 1, A and B). In a separate set of studies, mice were given mAb to V7 and immunized with F3. Elimination of V7⁺ cells did not abrogate the protective efficacy of F3. Both groups of F3-immunized mice contained significantly fewer (p < 0.01) CFU in lungs and spleens compared with controls (Fig. 1, C and D).

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Depletion of V6+ cells reverses the protective efficacy of F3 immunization. Mice (n = 6/group) were treated with mAb to V6 or with an equal mount of rat IgG and immunized with F3. Four weeks after immunization mice were challenged with 2.0 x 106 yeast cells i.n. Fungal recovery was assessed in lungs (A) and spleens (B) of mice at wk 1 of infection. In a second set of studies groups of F3-immunized mice (n = 6/group) were given either rat IgG or mAb to V7 and infected i.n. with 2.0 x 106 yeast cells. At wk 1 of infection, lungs (C) and spleens (D) were assessed for fungal burden. One of two experiments is shown. Data are the mean ± SEM. *, p < 0.01. E, Survival curve of mice immunized with F3 and treated with IgG (n = 8; ), mAb to V6 (n = 7; ), or mAb to V7 (n = 8; ). An additional set of controls (n = 8; •) was unimmunized. Mice were challenged with 1.25 x 107 yeast cells i.n., and survival was monitored for 40 days.

One explanation for the effect of the elimination of V6⁺ cells is that this family is pivotal for host defenses to Hc even in unimmunized mice. To address this issue, naive C57BL/6 mice were administered mAb to V6 or rat IgG and then infected i.n. with 2 x 10⁶ Hc yeasts. At wk 1 of infection, fungal recovery (mean log₁₀ CFU ± SEM) in lungs (6.60 ± 0.01 CFU) and spleens (4.95 ± 0.11 CFU) did not differ significantly (p > 0.05) from that in lungs (6.85 ± 0.06 CFU) and spleens (5.21 ± 0.08 CFU) of mice depleted of V6⁺ cells.

Adoptive transfer of protective cells from immunized mice

Once we established that V6⁺ cells were crucial for generation of an F3-reactive T cell line and for vaccine efficacy in vivo, a series of experiments was conducted to determine whether cells from F3-immunized mice could adoptively transfer protection into immunodeficient mice. Mice lacking TCR ⁺ cells were selected as recipients for evaluating the protective efficacy of adoptively transferred cells because they are susceptible to Hc, and T cells may repopulate their organs.

Splenocytes from F3- and BSA-immunized mice were harvested and stimulated in vitro with 20 µg/ml of Ag for 5 days, and cells were transferred to TCR ^-/- mice that were infected i.n. 8 h later. As an additional control, a group of TCR ^-/- mice received no cells. Transfer of the splenocytes from F3-immunized, but not BSA-immunized, mice reduced (p < 0.01) the fungal burden at wk 1 of infection (Fig. 2, A and B) and improved survival (p < 0.01; Fig. 2C). At the termination of the study the recipients of F3-stimulated splenocytes were sacrificed, and lungs and spleens were assessed for fungal burden. The mean log₁₀ CFU values (±SEM) in lungs and spleens were 2.77 ± 0.21 and 3.79 ± 0.20 CFU, respectively.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Transfer of splenocytes from F3-immunized mice confers protection in TCR -/- mice. Spleen cells from mice immunized with F3 or BSA were stimulated in vitro with 20 µg/ml Ag, respectively, for 4 days, and the cells were harvested and washed. Each mouse was inoculated i.v. with 2 x 107 splenocytes and 8 h later was infected with 2.0 x 106 yeast cells i.n. At wk 1 of infection, a group of mice (n = 6–7) was sacrificed, and CFU were enumerated in lungs (A) and spleens (B) of mice. Data are the mean ± SEM. In addition, mice (n = 6–7) were followed for survival (C). *, p < 0.01.

Elimination of V6⁺ cells from F3- immunized mice abrogates the protective activity of adoptively transferred cells

Mice were given rat IgG or mAb to V6 beginning 1 wk before vaccination with F3. Treatment with Ab was continued throughout the period of vaccination and extended for 2 wk after the last injection of F3. At that time spleen cells were harvested and stimulated with F3 for 5 days, and cells were injected i.v. into TCR ^-/- mice (2 x 10⁷ splenocytes/mouse). Eight hours later mice were infected i.n. with Hc yeasts. At 1 wk of infection, the lungs and spleens of mice were examined for fungal burden. The organs of TCR ^-/- mice that received F3-stimulated splenocytes contained significantly fewer CFU (p < 0.01) than those of mice that received no cells. Elimination of V6⁺ cells reversed the capacity of F3-stimulated splenocytes to mediate protection (Fig. 3, A and B). Transfer of splenocytes from F3-immunized mice promoted the survival of TCR ^-/- mice, whereas cells from V6⁺-depleted mice did not (Fig. 3C). The surviving mice were sacrificed, and CFU in lungs and spleens was assessed. The mean log₁₀ CFU was <10³ in both lungs and spleens.

View larger version (33K): [in this window] [in a new window]   FIGURE 3. Elimination of V6+ cells abrogates the capacity of splenocytes from F3-immunized mice to mediate protection. Spleen cells from mice administered mAb to V6 or rat IgG and immunized with F3 were stimulated in vitro with 20 µg/ml Ag for 4 days. Cells were harvested, and 2 x 107 were injected i.v. into TCR -/- mice. Eight hours later mice were infected with 2.0 x 106 yeasts i.n. At wk 1 of infection a group of mice (n = 6–7) was sacrificed, and CFU were enumerated in lungs (A) and spleens (B) of mice. Data are the mean ± SEM. In addition, mice (n = 6–7) were followed for survival (C). *, p < 0.01.

A V6⁺ T cell clone confers protection in TCR ^-/- mice

The T cell clone 9.14, which expresses V6, was injected i.v. into TCR ^-/- mice 8 h before i.n. challenge with Hc. Separate sets of mice received no cells; SOG, an OVA-reactive clone; or clone 2.6, a clone that expresses V14. Mice that did not receive cells and mice that received SOG or 2.6 all died by day 17 (Fig. 4). In contrast, 60% of recipients of 9.14 survived for 40 days (p < 0.01). After 40 days, the remaining mice were sacrificed, and the fungal burden in lungs and spleens was quantified. Both sets of organs contained <10³ CFU.

View larger version (17K): [in this window] [in a new window]   FIGURE 4. A V6+ T cell clone adoptively transfers protective immunity. TCR -/- mice were injected with 2 x 106 T cells from an OVA-reactive clone (SOG), an F3-reactive V14+ clone (2.6), or an F3-reactive V6+ clone (9.14) and then inoculated with 2.0 x 106 yeasts i.n. 8 h later. Mice (n = 5) were followed for survival.

Trafficking of the V6⁺ and V14⁺ clones in TCR ^-/- mice

We examined whether the in vivo survival of the V6⁺ clone differed from that of the VB14⁺ clone. TCR ^-/- mice were infected with Hc and injected with either clone. At 3 and 7 days postinfection lungs and spleens were examined for the presence of the clones by FACS. To determine the percentage of V6⁺ or V14⁺ cells, we gated on the CD3⁺ population. On day 3 postinfection the mean number and percentage of CD3⁺ V6⁺ cells in lungs and spleens significantly exceeded (p < 0.05) those of CD3⁺ V14⁺ cells in those organs (Table III). On day 7 postinfection the number and percentage of CD3⁺V6⁺ cells in lungs and spleens were higher than those of CD3⁺ V14⁺, but the data failed to reach statistical significance (p > 0.05; Table III). The data also indicate that between days 3 and 7 of infection there was expansion of each clonal population within lungs and spleens of mice.

View this table: [in this window] [in a new window]   Table III. In vivo detection of CD3+V6+ and CD3+V14+ cells in lungs and spleens of TCR-/- micea

Because the elimination of V6⁺ cells abrogated the protective immune response induced by F3, we sought to determine whether cytokine production was modulated by depletion of these cells. Four weeks after immunization with F3, splenocytes from mice that were treated with mAb to V6 or with rat IgG were stimulated in vitro with F3, and supernatants were harvested. Splenocytes from mice devoid of V6⁺ cells produced similar amounts (p > 0.05) of IFN-, TNF-, GM-CSF, IL-4, and IL-10 compared with mice given rat IgG.

Additional studies examined the cytokine production by clones 2.6 (V14⁺) and 9.14 (V6⁺). Clone 9.14 released substantially more IFN-, TNF-, and GM-CSF than clone 2.6 (Table IV). Production of IL-4 was negligible from both lines, and IL-10 generation was similar (Table IV). Analysis of six other V6⁺ clones revealed a cytokine profile similar to that of 9.14 (data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Accumulated evidence has documented clonal or oligoclonal expansion of V families in experimental models of infection and autoimmunity (12, 13, 14, 15, 16). Likewise, limited heterogeneity in TCR usage has been demonstrated in humans with some infectious diseases (17, 18), multiple sclerosis (19), and obliterative bronchioloitis associated with lung transplantation (20). In a high proportion of studies performed during experimental infection, associations have been established between disease and TCR bias, but little is known about the role of the expanded V family in disease recovery or exacerbation. One prominent example has been a model of cutaneous leishmaniasis in which the same TCR family (V8-V4) emerges in both susceptible and resistant mice (12). These results strongly suggest that the same T cell family contributes to clearance or progression of the pathogen depending on the genetic background of the host.

The predominance of V6⁺ T cell clones provided an impetus to analyze clonal usage in F3-immunized mice that were depleted of V6⁺ cells. We postulated that V14⁺ T cell clones would emerge from these mice, because this family was detected in F3-immunized animals. Multiple attempts to generate clones from V6⁺ cell-depleted mice failed. The inability of V6⁺ cell-depleted mice to generate T cell clones was associated with a nonexistent response to F3. Similarly, mAb-mediated elimination in DBA/2 mice of the dominant TCR family, V8.2⁺ cells, that responds to peptide 110–121 from sperm whale myoglobin results in a weak in vitro response to Ag by cells from these mice (21). In both experimental systems a single V family was the key to Ag responsiveness.

We recently reported that in the lungs of C57BL/6 mice with secondary histoplasmosis, the V6⁺ cell is the only V family to undergo expansion (9). This population is significantly elevated on days 7, 10, and 14 of infection and declines thereafter. Depletion of these cells exacerbates the course of pulmonary Hc (9). The pivotal contribution of this cell population raised the possibility that an epitope from F3 may have driven the expansion of V6⁺ cells in the lungs of Hc-infected mice. Comparative analysis of the CDR3 sequences found in the F3-derived clones and the V6⁺ cells from the lungs of mice with secondary infection reveal no overriding similarities. It is unlikely that the V6⁺ family from the lungs of infected mice is recognizing the same Ag.

The finding that only two V families were detected among 28 F3-reactive clones was unexpected, because we had postulated that TCR usage would be more diverse given the relatively large size of the polypeptide. The preferential usage of J families and CDR3 expression in conjunction with the outgrowth of a fairly limited set of V families strongly suggests that the response is driven by a peptide or, at the very least, a limited set of peptides. Identification of the peptide(s) from this protein that drives the expansion of V6 and/or V14 families is underway.

Insight into the functional role of V6⁺ cells in vaccine-induced immunity was accomplished in two different sets of experiments. Elimination of V6⁺ cells reversed the protective activity of F3 in intact mice. In contrast, V6⁺ cells were dispensable for primary infection of mice. Thus, the remaining T cells from F3-immunized mice that were depleted of V6⁺ cells failed to compensate for the abolished population.

We complemented the above experiments with a companion set of studies that sought to determine whether V6⁺ cells were necessary for protection by adoptive transfer. Splenocytes from mice immunized with F3 and treated with mAb to V6 did not transfer protection in TCR ^-/- mice, as measured by a reduction in CFU and survival. To validate this finding, transfer of a V6⁺, but not V14⁺, T cell clone conferred protection despite the similarity in the CDR3 sequence between the two clones. The results indicate that V6⁺ cells are critical in vaccine-induced immunity.

One concern in using T cell clones is that they may not traffic normally in an intact host (21, 22). The likely mechanism is that these cells do not express CD62L (MEL-14 or L-selectin), which is required for lymphocyte homing to peripheral lymphoid organs (23, 24). To determine whether the protective efficacy of the V6⁺ clone was a result of improved survival within the host, we examined the lungs and spleens of Hc-infected TCR ^-/- mice for the presence of V6⁺ or V14⁺ cells. Interestingly, the percentage and number of V6⁺ cells in each organ exceeded those of V14⁺ cells, and the differences were statistically significant on day 3 postinfection. Hence, the protective effect exerted by CD3⁺V6⁺ cells may be explained in part by the elevated numbers of this population found in lungs and spleens during the early phase of infection (3 days).

In experimental infection with viable Hc yeasts, IFN-, TNF-, and GM-CSF are critical for effective host resistance to this fungus (6, 7, 25, 26, 27, 28, 29, 30, 31). By contrast, IL-4 and IL-10 antagonize host defenses (25, 26, 31). We sought to determine whether the ineffectiveness of cells from F3-immunized mice administered mAb to V6 to confer protection was associated with alterations in cytokine generation. Splenocytes from V6⁺ cell-depleted mice immunized with F3 produced nearly equal amounts of cytokines as cells from immunized mice with V6⁺ cells, but the former did not proliferate in response to Ag. The dissociation between cytokine production and inability to proliferate was unexpected. Others (32, 33) have reported that proliferation and cytokine release are not inextricably connected. The most likely explanation is that the TCR signaling necessary for proliferation differs from that necessary for cytokine generation. We could not discriminate between the two populations of cells by in vitro generation of cytokines that are known to influence the course of infection with Hc. Hence, it is possible that another cytokine or chemokine that potentially contributes to host resistance is released only by V6⁺ cells.

When cytokine production by T cell clones was analyzed, there was a correlation between protective efficacy and release of cytokines that augment host resistance. Collectively, these findings indicate that the protective efficacy of unfractionated spleen cells may be more dependent on the ability to undergo clonal expansion. At the clonal level, cytokine production discriminated between a protective and a nonprotective clone.

In summary, we have demonstrated a pronounced bias in the TCR repertoire to a protective immunogen from the pathogenic fungus, Hc. V6⁺ cells were instrumental in proliferation to this Ag as well as in protective efficacy. These results demonstrate that a single V family may be requisite for successful vaccination to a polypeptide.

	Footnotes

 
¹ This work was supported by Grants AI42747 and AI34361 from the National Institutes of Health and a Merit Review Grant from Veterans Affairs.

² Address correspondence and reprint requests to Dr. George S. Deepe, Jr., Division of Infectious Diseases, Department of Medicine, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0560. E-mail address: george.deepe{at}uc.edu

³ Abbreviations used in this paper: Hc, Histoplasma capsulatum; hsp, heat shock protein; i.n., intranasal(ly).

Received for publication January 31, 2001. Accepted for publication June 7, 2001.

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