A Deficiency in Gamma Interferon or Interleukin-10 Modulates T-Cell-Dependent Responses to Heat Shock Protein 60 from Histoplasma capsulatum
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感染与免疫杂志 2005年第4期
Department of Molecular Genetics, Biochemistry, and Microbiology
Division of Infectious Diseases
Veterans Affairs Hospital, University of Cincinnati College of Medicine, Cincinnati, Ohio
ABSTRACT
Immunization of mice with heat shock protein 60 from Histoplasma capsulatum or a polypeptide from the protein designated F3 confers protection. V8.1/8.2+ T cells are critically important for the protective efficacy of this antigen. The production of interleukin-10 and gamma interferon following vaccination is essential for efficacy. In this study, we sought to determine whether the absence of either cytokine modified the repertoire of antigen-reactive T cells and whether it altered the functional properties of T cells. Mice lacking gamma interferon or interleukin-10 manifested a skewed repertoire compared to that of wild-type mice. The bias was most marked in gamma interferon-deficient mice and modestly altered in interleukin-10-deficient animals. The altered repertoire in gamma interferon-deficient mice could not be explained at the level of antigen presentation or by the absence of this population from mice. The proportion of T cells from interleukin-10-deficient mice manifesting a Th1 phenotype was greatly increased compared to that from wild-type animals. Transfer of splenocytes from gamma interferon- or interleukin-10-deficient mice immunized with heat shock protein 60 failed to confer protection in T-cell receptor /–/– mice. The transfer of T-cell clones that did not produce both cytokines failed to prolong survival in T-cell receptor /–/– mice, whereas the clones with the same features that were derived from wild-type mice did. These results indicate that the cytokine milieu influences the shape of the T-cell receptor repertoire and support the importance of gamma interferon and interleukin-10 in the efficacy of heat shock protein 60.
INTRODUCTION
Infection with the dimorphic, pathogenic fungus Histoplasma capsulatum causes a wide spectrum of disease in humans. Illness ranges from mild influenza-like symptoms to life-threatening progressive infection associated with disturbances in coagulation. Infection is initiated once the inhaled conidia convert into the yeast phase. H. capsulatum yeasts are readily phagocytosed by resident macrophages, in which they survive and replicate until cellular immunity, in particular, T-cell-mediated immunity, is activated to limit their intracellular growth (6). Thus, immunity against H. capsulatum relies largely, if not exclusively, on the generation of an effective adaptive immune response characterized by a collaboration between professional phagocytes, including dendritic cells and T cells (2, 15, 25, 26, 30, 31).
We have previously demonstrated that immunization with H. capsulatum heat shock protein 60 (hsp 60) or a domain within it that is termed F3 confers a protective response in mice challenged with nonlethal or lethal inocula (7, 16, 18). The efficacy of this immunogen is dependent on the presence of CD4+ T cells and endogenous gamma interferon (IFN-), interleukin 12 (IL-12), and IL-10 (8). Subsequent analysis of the T-cell receptor (TCR) repertoire in C57BL/6 mice has demonstrated that CD4+ V8.1/8.2+ cells are preferentially expanded following immunization with recombinant hsp 60 (rhsp 60) and are essential for the generation of protective immunity to this antigen (27). Elimination of this subpopulation of T cells blunts vaccine-induced immunity. Among V8.1/8.2+ T-cell clones, only the population that produces IFN- and responds to the protective fragment from hsp 60 confers protection upon adoptive transfer into TCR /–/– mice (27).
The cellular and molecular factors that are important in immunity associated with vaccination with rhsp 60 have been identified independently (8, 27). To date, we have not explored the question of whether the production of cytokines is intricately linked to the development of the TCR repertoire and the generation of protective immunity following immunization with rhsp 60. The results demonstrate that mice lacking IFN- or IL-10 manifest an altered TCR repertoire and that T cells from mice lacking either cytokine exhibit altered T-cell effector functions compared to those of cells from wild-type (WT) animals.
MATERIALS AND METHODS
Animals. Male C57BL/6 IL-10–/–, IFN-–/–, and TCR /–/– mice were purchased from the Jackson Laboratories, Bar Harbor, Maine. Mice were used when they were between 6 and 8 weeks of age. Animals were housed under barrier conditions and were age and sex matched for all experiments. All animal experiments were done in accordance with the Animal Welfare Act guidelines of the National Institutes of Health.
H. capsulatum culture and infection of mice. H. capsulatum strain G217B yeast cells were cultured and quantified as described previously (12). Following anesthesia with isofluorane, mice were infected intranasally with H. capsulatum. Mice were challenged with 5 x 105 or 2 x 106 yeasts in approximately 35 μl of Hanks balanced salt solution (HBSS).
Production of rhsp 60 and F3. rhsp 60 and F3 from H. capsulatum were generated as described previously (7, 16). Each protein contained less than 10 pg of endotoxin/μg of protein.
Splenocyte preparation. Splenocytes were obtained by teasing spleens apart between the frosted ends of two glass slides. Cells were washed three times with HBSS and resuspended in RPMI 1640 (Biowhittaker, Walkersville, Md.) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 1% sodium pyruvate, 1% nonessential amino acids, 5 x 10–5 M 2-mercaptoethanol, and 10 μg of gentamicin per ml if they were used for the establishment or expansion of T-cell lines and clones but with only 10% FBS, gentamicin, and L-glutamine if they were used in proliferation assays.
Generation of T-cell lines and clones. Mice were immunized with 100 μg of rhsp 60 subcutaneously and again after a 2-week interval. Spleen cells were harvested 2 weeks after the final immunization and stimulated with rhsp 60 at 5 μg/ml for 2 weeks in supplemented RPMI 1640. Following the initial stimulation, fresh media and antigen were added at 2-week intervals with fresh irradiated spleen cells that served as antigen-presenting cells. Individual T-cell clones were obtained by limiting dilution following several passages of the parent line and propagated as described above with the addition of 5% supernatant derived from rat splenocytes stimulated with concanavalin A (Sigma-Aldrich, St. Louis, Mo.) for 48 h (9, 11).
Proliferation assay. To each well of a microtiter plate, 2 x 104 resting T cells in 0.1 ml of RPMI 1640 containing 10% FBS, 10 μg of gentamicin/ml, and 5 x 105 irradiated splenocytes in 0.1 ml of medium were added; 50 μl of either medium or antigen (F3 or rhsp 60) was added to each well (the final concentration in each well was 5 μg/ml). The cells were incubated for 72 h. Cells were pulsed 18 h before being harvested with the addition of 1 μCi of [3H]thymidine (specific activity, 6.7 Ci/mmol; New England Nuclear, Boston, Mass.). Cells were harvested to glass fiber filter paper, and the incorporation of radioactivity was measured by a liquid scintillation counter. Activity was analyzed as the stimulation index: the counts per minute of cells stimulated with antigen divided by the counts per minute of cells in media alone. Reactivity to rhsp 60 or F3 was defined as a stimulation index indicating a 2.5-fold increase in the proliferation of cells compared to that of cells that were cultured in medium alone.
V analysis. Expression of the V chain of T-cell clones was assessed by reverse transcription-PCR (RT-PCR) and confirmed by flow cytometry. Briefly, RNA was isolated from the T cells using RNAzol (Biotecx Laboratories, Houston, Tex.) 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 constant region of the chain of the TCR and incubating the RNA with avian myeloblastosis virus reverse transcriptase (Invitrogen, La Jolla, Calif.). PCR was performed with a C antisense primer and a sense primer specific for each of the V families. The amplified cDNA from each T cell was electrophoresed in a 1% agarose gel, transferred to a nylon membrane, and incubated with a digoxigenin-labeled probe specific for the C region as described previously (17).
For flow cytometric analysis, T cells (105 cells) were incubated with the appropriate biotinylated monoclonal antibody to the V family (BD PharMingen, San Diego, Calif.) for 15 min at 4°C. After being washed three times, the cells were incubated with streptavidin-phycoerythrin for 15 min at 4°C and washed three times. Cells were then fixed with 1% paraformaldehyde. Fluorescence was measured using a FACScaliber flow cytometer. V expression was determined by examining the number of cells within the phycoerythrin channel divided by the total number of cells counted.
Analysis of cytokine production. Cytokine production was measured using commercially available enzyme-linked immunosorbent assay kits for IFN-, IL-4 (Pierce-Endogen, Rockford, Ill.), and IL-10 (BD Pharmingen). Cytokine measurements are presented as the amount of cytokine produced following stimulation with correction for the levels of production from unstimulated cells.
Quantitative organ culture. Mice were sacrificed and examined for fungal burdens in lungs and spleens (17). Organs were harvested and homogenized in 5 ml of 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 numbers of CFU per organ ± standard errors of the means.
Adoptive transfer. Numbers of splenocytes or T-cell clones were expanded to generate sufficient cells to inject intravenously into each TCR /–/– mouse. Mice were injected with either 2 x 107 splenocytes or 2 x 106 T cells and then infected with 5 x 105 H. capsulatum yeasts intranasally 8 h later.
Statistical analysis. The chi-square test or z test was used to compare differences between groups. The log rank sum test was employed to analyze differences in rates of survival. Significance was achieved at a P of <0.05.
RESULTS
V expression of rhsp 60-reactive T-cell clones. Since endogenous IFN- and IL-10 were critically important to the protective efficacy of rhsp 60 (8), we sought to determine if the absence of either of these cytokines altered the repertoire of T cells at the clonal level. Groups of WT, IFN-–/–, and IL-10–/– mice were immunized with 100 μg of rhsp 60 twice with a 2-week separation. Analysis of V expression of each T-cell clone was determined by RT-PCR in conjunction with flow cytometry. T-cell clones derived from IFN-–/– and IL-10–/– mice following immunization with rhsp 60 demonstrated an altered repertoire compared to that of WT mice. V8.1/8.2+ cells were substantially reduced in IL-10–/– (P < 0.05) and IFN-–/– (P < 0.01) mice compared to numbers in WT mice (Fig. 1). The decrement in the proportion of V8.1/8.2+ clones could not be explained by a diminished number of this cell population in the spleens of IFN-–/– or IL-10–/– mice. By flow cytometry, the proportion of CD3+V8.1/8.2+ cells in the spleens of WT mice (21% ± 2%; n = 4) was not different from the proportion in the spleens of IL-10–/– (19% ± 2%; n = 4) or IFN-–/– (24% ± 2%; n = 4) mice.
The proportions of monoclonal V4+ and V6+ cells derived from either IL-10–/– or IFN-–/– mice did not differ from proportions in WT mice. There were differences in the levels of expression of V7, V11, and V14, but the numbers of clones were too small to analyze them statistically in a meaningful way.
Cytokine profile among rhsp 60-reactive T-cell clones. The production of Th1-type cytokines is critically important in host resistance to H. capsulatum and for the efficacy of rhsp 60 (1, 8, 29, 32). Therefore, we examined cytokine production by T-cell clones from WT, IL-10–/–, and IFN-–/– mice to determine the effector capacities of these cells. Clones were incubated with or without 5 μg of rhsp 60 per ml, and supernatants were harvested after 48 h and analyzed by enzyme-linked immunosorbent assay. Nearly equal proportions of clones from WT and IL-10–/– mice were classified as having the Th1 phenotype by virtue of producing IFN- (Fig. 2A). However, if the analysis was restricted only to V8.1/8.2+ cells, 90% of the clones from IL-10–/– mice were Th1, whereas only 50% of those from WT mice were of this phenotype. We also examined IL-10 production by clones from WT and IFN-–/– mice. Nearly all clones (90%) from WT mice produced IL-10, and a much smaller proportion (37%) released this cytokine from IFN-–/– mice (Fig. 2C). If V8.1/8.2+ cells were analyzed, the disparity in proportions was similar to that found for all clones (Fig. 2D). These data demonstrate that the absence of cytokines skewed not only the TCR profile but also the generation of critically important cytokines by T cells.
Reactivity to F3. The protective domain of rhsp 60 spans amino acids 172 to 443 (7). Immunization with polypeptide induces a protective immune response in mice. Previously, we demonstrated that among V8.1/8.2+ monoclonal T cells from C57BL/6 mice, recognition of this polypeptide was another determinant that distinguished a protective T cell from a nonprotective one (27). Hence, we assessed reactivity among our clones to it. The proportion of all clones recognizing F3 was markedly diminished (P < 0.01) among those derived from IFN-–/– mice and less so (P < 0.05) from IL-10–/– mice (Fig. 2E). A similar profile was true if only V8.1/8.2+ cells were analyzed (Fig. 2F).
Is the alteration in TCR usage a result of defective antigen presentation To examine whether deficient animals demonstrated altered antigen presentation, splenocytes from wild-type and cytokine-deficient animals were harvested and irradiated for use as antigen-presenting cells. Irradiated splenocytes from IFN-–/– mice manifested an ability to stimulate a V8.1/8.2+ clone similar to that of antigen-presenting cells from WT mice (Fig. 3). Data for one concentration of rhsp 60, 5 μg/ml, are shown. Similar findings were noted with 10 and 1 μg/ml (data not shown).
Protection-mediated rhsp 60 at the single-cell level is dependent on the coexpression of IFN- and IL-10. WT, IFN-–/–, and IL-10–/– mice were twice immunized at an interval of 2 weeks with rhsp 60. Splenocytes from immunized mice were transferred into TCR /–/– mice that were infected with 5 x 105 yeasts and monitored for survival. Mice receiving cells from WT animals manifested a prolonged survival compared to that of each of the other groups (Fig. 4A). Cultures of lungs and spleens at the termination of the experiment revealed that the organs of the surviving mice contained no detectable H. capsulatum CFU.
We previously demonstrated that the protective V8.1/8.2+ T cells must be reactive to F3 and produce IFN- (27). The clone that we utilized for those experiments also produced IL-10 (26). Thus, we sought to extend those studies and determine if coexpression of IL-10 and IFN- in conjunction with F3 reactivity was necessary to confer protection. To accomplish this, we utilized a V8.1/8.2+ T-cell clone that reacted to F3 that was derived from IL-10–/– mice (clone 6b) and clone D, which was the original clone derived from WT mice (26). In addition, another V8.1/8.2+ T-cell clone (clone 6) that coexpressed IL-10 and IFN- and reacted to F3 was used. T cells were transferred into TCR /–/– mice, and their survival was monitored. As seen in Fig. 4B, only clones that secreted both IL-10 and IFN- significantly (P < 0.01) prolonged survival.
DISCUSSION
rhsp 60 is a protective antigen that stimulates an increased production of IFN-, IL-10, and IL-12 by mouse splenocytes and causes the expansion of murine T cells that predominantly express V8.1/8.2 (8, 27). More importantly, subsets of V8.1/8.2+ T cells that are F3 reactive and produce IFN- are able to confer a protective response to T-cell-deficient mice (27). Thus, defined populations of T cells are involved in the generation of the protective response to rhsp 60. In results presented herein, we explored the possibility that the absence of IFN- or IL-10 would modulate the T-cell response to rhsp 60. We demonstrated that, indeed, the a lack of either one altered the frequency of T cells expressing V8.1/8.2, the cytokine profile of these T cells, and the proportion that reacted to the protective domain of this antigen. The results were more profound in mice that lacked IFN-. These findings indicate that the presence or absence of a cytokine necessary for protection has effects beyond the mere absence of that cytokine. The repertoire of T cells which the host utilizes to combat infection is in part shaped by the cytokine environment.
Among the alterations associated with the absence of IFN- was the marked decrement in the proportion of V8.1/8.2+ cells that are key in the generation of vaccine-induced immunity (27). One explanation for the altered TCR repertoire generated in the absence of IFN- is a defect in antigen presentation. IFN- regulates a number of effects on antigen presentation. These effects include increases in class I and class II major histocompatibility complex antigens, induction of lysosomal cathepsins that are important in the generation of immunogenic peptides, and induction of the proteosome pathway (4). This explanation appears to be unlikely since antigen-presenting cells from the IFN-–/– mice were as efficient as those from WT mice in stimulating T-cell responses. Alternatively, the altered TCR may have been a result of fewer V8.1/8.2+ cells in the spleens of animals, but this was not the case. The failure of this T-cell population to expand following vaccination in IFN-–/– as well as IL-10–/– mice remains unknown. The results unequivocally demonstrate that the presence or absence of cytokines may shape the TCR repertoire to an antigen or antigens.
Cytokine production among all T-cell clones was altered compared to that in cells from WT animals. In IL-10–/– mice, the proportion of Th1 cells was nearly 100% of that in WT animals. The absence of IL-10 greatly influenced the generation of Th1 cells, as has been observed previously. In mice, the absence of IL-10 or the blockade of its cognate receptor enhances the protective immune response to Leishmania donovani, Plasmodium spp., Listeria monocytogenes, Coccidioides immitis, Mycobacterium spp., and H. capsulatum (10, 13, 20, 23, 24, 28). Despite the pronounced skewing of the immune response to a Th1 phenotype, neither polyclonal nor monoclonal cells from IL-10–/– mice induced protection in TCR /–/– mice.
Additional analysis revealed that the proportion of T-cell clones that reacted to the protective domain of rhsp 60, F3, was decreased compared to that in WT mice whether all clones were examined or whether the examination was restricted to V8.1/8.2+ cells. Since responsiveness to this polypeptide is an important functional feature of protective T cells, the results indicate that IFN- and IL-10 are important for the development of this population. The failure of clone 6b, which did not produce IL-10, to confer protection may be a result the absence of this cytokine in the antifungal effector pathway. Other explanations should be considered. It is possible that clones that do not produce IL-10 are unable to traffic in a manner similar to that of clones that do release IL-10. In addition, the in vivo survival of clones that lack IL-10 may be impaired compared to that of IL-10+ clones. Nevertheless, our results demonstrate that the cytokine environment has a major impact on the type of T cells that emerge following immunization with a single protein.
The importance of IFN- and IL-10 in response to a pathogen has been documented in several models of infection. In most cases, the production of IL-10 or IFN- serves to inhibit the production of the other. This balance is vital in maintaining the integrity of the immune response without generating immunopathology at the site of infection. In our vaccination model, however, the presence of IL-10 may be necessary to obviate a massive inflammatory response associated with Th1 cells and to confer protection that is unrelated by upregulating antifungal effectors (22). The requirement for IL-10 in the generation of a protective immune response is uncommon. This cytokine is essential for mice infected with L. major to maintain protective immunity, principally because its presence allows low numbers of parasites to persist. Their persistence sustains the protective immune response (3). In addition, IL-10 has been reported to be important for the enhanced protection associated with pneumococcal polysaccharide vaccine and for several tumor vaccines (14, 19, 21).
The importance of endogenous IL-10 for the optimal expression of immunity mediated by rhsp 60 is linked to the production of IFN-. Only dual-expression clones that also reacted to F3 were protective. The mechanisms by which these two cytokines interact to trigger a protective response by a population of T cells are unclear. Production of IL-10 has been shown to limit the apoptosis of antigen-presenting cells and thus extend the time of antigen processing and presentation (5). Delivery of this cytokine by T cells may enhance the efficiency of antigen processing and prolong the time in which antigens may be recognized. Moreover, it is possible that the production of IL-10 by T cells may protect them from apoptosis and thus extend their functional life spans. Accordingly, the ability of IFN-+ IL-10+ T cells to promote immunity may exceed that of IFN-+ IL-10– cells. Work is under way to test this hypothesis.
We previously reported that the major source of IL-10 in splenocytes from vaccinated mice was not of T-cell origin (8). However, it is evident that T cells are a source—if not the major source. The findings suggest that analysis of polyclonal populations is not sufficiently sensitive to locate critically important T-cell populations that function to protect.
In survival studies, the transfer of cells from WT mice immunized with rhsp 60 conferred sterilizing immunity, whereas the transfer of a T-cell clone even though it was a Th1 clone did not induce the same type of protection. Although mice that received a protective clone exhibited prolonged survival, sterilizing immunity was not achieved. The most likely explanation for this finding is that protective immunity relies upon the interaction with other T-cell families. Thus, a clonal population may not be sufficient in a mouse that lacks all T cells that bear the / receptor.
In summary, we have demonstrated that the absence of IFN- and, to a lesser extent, IL-10 influences a number of critical markers of protective T cells that are generated in response to vaccination with rhsp 60. These findings have implications for T-cell vaccines that rely predominantly if not exclusively on T cells for combating the infectious process.
ACKNOWLEDGMENTS
This work was supported by grants AI34361 and AI42747 from the National Institutes of Health and by a Merit Review grant from the Department of Veterans Affairs.
We thank Reta Gibbons for technical assistance.
Present address: Tupper Research Institute, Division of Geographic Medicine and Infectious Diseases, New England Medical Center, Tufts University School of Medicine, Boston, MA 02111.
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Division of Infectious Diseases
Veterans Affairs Hospital, University of Cincinnati College of Medicine, Cincinnati, Ohio
ABSTRACT
Immunization of mice with heat shock protein 60 from Histoplasma capsulatum or a polypeptide from the protein designated F3 confers protection. V8.1/8.2+ T cells are critically important for the protective efficacy of this antigen. The production of interleukin-10 and gamma interferon following vaccination is essential for efficacy. In this study, we sought to determine whether the absence of either cytokine modified the repertoire of antigen-reactive T cells and whether it altered the functional properties of T cells. Mice lacking gamma interferon or interleukin-10 manifested a skewed repertoire compared to that of wild-type mice. The bias was most marked in gamma interferon-deficient mice and modestly altered in interleukin-10-deficient animals. The altered repertoire in gamma interferon-deficient mice could not be explained at the level of antigen presentation or by the absence of this population from mice. The proportion of T cells from interleukin-10-deficient mice manifesting a Th1 phenotype was greatly increased compared to that from wild-type animals. Transfer of splenocytes from gamma interferon- or interleukin-10-deficient mice immunized with heat shock protein 60 failed to confer protection in T-cell receptor /–/– mice. The transfer of T-cell clones that did not produce both cytokines failed to prolong survival in T-cell receptor /–/– mice, whereas the clones with the same features that were derived from wild-type mice did. These results indicate that the cytokine milieu influences the shape of the T-cell receptor repertoire and support the importance of gamma interferon and interleukin-10 in the efficacy of heat shock protein 60.
INTRODUCTION
Infection with the dimorphic, pathogenic fungus Histoplasma capsulatum causes a wide spectrum of disease in humans. Illness ranges from mild influenza-like symptoms to life-threatening progressive infection associated with disturbances in coagulation. Infection is initiated once the inhaled conidia convert into the yeast phase. H. capsulatum yeasts are readily phagocytosed by resident macrophages, in which they survive and replicate until cellular immunity, in particular, T-cell-mediated immunity, is activated to limit their intracellular growth (6). Thus, immunity against H. capsulatum relies largely, if not exclusively, on the generation of an effective adaptive immune response characterized by a collaboration between professional phagocytes, including dendritic cells and T cells (2, 15, 25, 26, 30, 31).
We have previously demonstrated that immunization with H. capsulatum heat shock protein 60 (hsp 60) or a domain within it that is termed F3 confers a protective response in mice challenged with nonlethal or lethal inocula (7, 16, 18). The efficacy of this immunogen is dependent on the presence of CD4+ T cells and endogenous gamma interferon (IFN-), interleukin 12 (IL-12), and IL-10 (8). Subsequent analysis of the T-cell receptor (TCR) repertoire in C57BL/6 mice has demonstrated that CD4+ V8.1/8.2+ cells are preferentially expanded following immunization with recombinant hsp 60 (rhsp 60) and are essential for the generation of protective immunity to this antigen (27). Elimination of this subpopulation of T cells blunts vaccine-induced immunity. Among V8.1/8.2+ T-cell clones, only the population that produces IFN- and responds to the protective fragment from hsp 60 confers protection upon adoptive transfer into TCR /–/– mice (27).
The cellular and molecular factors that are important in immunity associated with vaccination with rhsp 60 have been identified independently (8, 27). To date, we have not explored the question of whether the production of cytokines is intricately linked to the development of the TCR repertoire and the generation of protective immunity following immunization with rhsp 60. The results demonstrate that mice lacking IFN- or IL-10 manifest an altered TCR repertoire and that T cells from mice lacking either cytokine exhibit altered T-cell effector functions compared to those of cells from wild-type (WT) animals.
MATERIALS AND METHODS
Animals. Male C57BL/6 IL-10–/–, IFN-–/–, and TCR /–/– mice were purchased from the Jackson Laboratories, Bar Harbor, Maine. Mice were used when they were between 6 and 8 weeks of age. Animals were housed under barrier conditions and were age and sex matched for all experiments. All animal experiments were done in accordance with the Animal Welfare Act guidelines of the National Institutes of Health.
H. capsulatum culture and infection of mice. H. capsulatum strain G217B yeast cells were cultured and quantified as described previously (12). Following anesthesia with isofluorane, mice were infected intranasally with H. capsulatum. Mice were challenged with 5 x 105 or 2 x 106 yeasts in approximately 35 μl of Hanks balanced salt solution (HBSS).
Production of rhsp 60 and F3. rhsp 60 and F3 from H. capsulatum were generated as described previously (7, 16). Each protein contained less than 10 pg of endotoxin/μg of protein.
Splenocyte preparation. Splenocytes were obtained by teasing spleens apart between the frosted ends of two glass slides. Cells were washed three times with HBSS and resuspended in RPMI 1640 (Biowhittaker, Walkersville, Md.) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 1% sodium pyruvate, 1% nonessential amino acids, 5 x 10–5 M 2-mercaptoethanol, and 10 μg of gentamicin per ml if they were used for the establishment or expansion of T-cell lines and clones but with only 10% FBS, gentamicin, and L-glutamine if they were used in proliferation assays.
Generation of T-cell lines and clones. Mice were immunized with 100 μg of rhsp 60 subcutaneously and again after a 2-week interval. Spleen cells were harvested 2 weeks after the final immunization and stimulated with rhsp 60 at 5 μg/ml for 2 weeks in supplemented RPMI 1640. Following the initial stimulation, fresh media and antigen were added at 2-week intervals with fresh irradiated spleen cells that served as antigen-presenting cells. Individual T-cell clones were obtained by limiting dilution following several passages of the parent line and propagated as described above with the addition of 5% supernatant derived from rat splenocytes stimulated with concanavalin A (Sigma-Aldrich, St. Louis, Mo.) for 48 h (9, 11).
Proliferation assay. To each well of a microtiter plate, 2 x 104 resting T cells in 0.1 ml of RPMI 1640 containing 10% FBS, 10 μg of gentamicin/ml, and 5 x 105 irradiated splenocytes in 0.1 ml of medium were added; 50 μl of either medium or antigen (F3 or rhsp 60) was added to each well (the final concentration in each well was 5 μg/ml). The cells were incubated for 72 h. Cells were pulsed 18 h before being harvested with the addition of 1 μCi of [3H]thymidine (specific activity, 6.7 Ci/mmol; New England Nuclear, Boston, Mass.). Cells were harvested to glass fiber filter paper, and the incorporation of radioactivity was measured by a liquid scintillation counter. Activity was analyzed as the stimulation index: the counts per minute of cells stimulated with antigen divided by the counts per minute of cells in media alone. Reactivity to rhsp 60 or F3 was defined as a stimulation index indicating a 2.5-fold increase in the proliferation of cells compared to that of cells that were cultured in medium alone.
V analysis. Expression of the V chain of T-cell clones was assessed by reverse transcription-PCR (RT-PCR) and confirmed by flow cytometry. Briefly, RNA was isolated from the T cells using RNAzol (Biotecx Laboratories, Houston, Tex.) 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 constant region of the chain of the TCR and incubating the RNA with avian myeloblastosis virus reverse transcriptase (Invitrogen, La Jolla, Calif.). PCR was performed with a C antisense primer and a sense primer specific for each of the V families. The amplified cDNA from each T cell was electrophoresed in a 1% agarose gel, transferred to a nylon membrane, and incubated with a digoxigenin-labeled probe specific for the C region as described previously (17).
For flow cytometric analysis, T cells (105 cells) were incubated with the appropriate biotinylated monoclonal antibody to the V family (BD PharMingen, San Diego, Calif.) for 15 min at 4°C. After being washed three times, the cells were incubated with streptavidin-phycoerythrin for 15 min at 4°C and washed three times. Cells were then fixed with 1% paraformaldehyde. Fluorescence was measured using a FACScaliber flow cytometer. V expression was determined by examining the number of cells within the phycoerythrin channel divided by the total number of cells counted.
Analysis of cytokine production. Cytokine production was measured using commercially available enzyme-linked immunosorbent assay kits for IFN-, IL-4 (Pierce-Endogen, Rockford, Ill.), and IL-10 (BD Pharmingen). Cytokine measurements are presented as the amount of cytokine produced following stimulation with correction for the levels of production from unstimulated cells.
Quantitative organ culture. Mice were sacrificed and examined for fungal burdens in lungs and spleens (17). Organs were harvested and homogenized in 5 ml of 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 numbers of CFU per organ ± standard errors of the means.
Adoptive transfer. Numbers of splenocytes or T-cell clones were expanded to generate sufficient cells to inject intravenously into each TCR /–/– mouse. Mice were injected with either 2 x 107 splenocytes or 2 x 106 T cells and then infected with 5 x 105 H. capsulatum yeasts intranasally 8 h later.
Statistical analysis. The chi-square test or z test was used to compare differences between groups. The log rank sum test was employed to analyze differences in rates of survival. Significance was achieved at a P of <0.05.
RESULTS
V expression of rhsp 60-reactive T-cell clones. Since endogenous IFN- and IL-10 were critically important to the protective efficacy of rhsp 60 (8), we sought to determine if the absence of either of these cytokines altered the repertoire of T cells at the clonal level. Groups of WT, IFN-–/–, and IL-10–/– mice were immunized with 100 μg of rhsp 60 twice with a 2-week separation. Analysis of V expression of each T-cell clone was determined by RT-PCR in conjunction with flow cytometry. T-cell clones derived from IFN-–/– and IL-10–/– mice following immunization with rhsp 60 demonstrated an altered repertoire compared to that of WT mice. V8.1/8.2+ cells were substantially reduced in IL-10–/– (P < 0.05) and IFN-–/– (P < 0.01) mice compared to numbers in WT mice (Fig. 1). The decrement in the proportion of V8.1/8.2+ clones could not be explained by a diminished number of this cell population in the spleens of IFN-–/– or IL-10–/– mice. By flow cytometry, the proportion of CD3+V8.1/8.2+ cells in the spleens of WT mice (21% ± 2%; n = 4) was not different from the proportion in the spleens of IL-10–/– (19% ± 2%; n = 4) or IFN-–/– (24% ± 2%; n = 4) mice.
The proportions of monoclonal V4+ and V6+ cells derived from either IL-10–/– or IFN-–/– mice did not differ from proportions in WT mice. There were differences in the levels of expression of V7, V11, and V14, but the numbers of clones were too small to analyze them statistically in a meaningful way.
Cytokine profile among rhsp 60-reactive T-cell clones. The production of Th1-type cytokines is critically important in host resistance to H. capsulatum and for the efficacy of rhsp 60 (1, 8, 29, 32). Therefore, we examined cytokine production by T-cell clones from WT, IL-10–/–, and IFN-–/– mice to determine the effector capacities of these cells. Clones were incubated with or without 5 μg of rhsp 60 per ml, and supernatants were harvested after 48 h and analyzed by enzyme-linked immunosorbent assay. Nearly equal proportions of clones from WT and IL-10–/– mice were classified as having the Th1 phenotype by virtue of producing IFN- (Fig. 2A). However, if the analysis was restricted only to V8.1/8.2+ cells, 90% of the clones from IL-10–/– mice were Th1, whereas only 50% of those from WT mice were of this phenotype. We also examined IL-10 production by clones from WT and IFN-–/– mice. Nearly all clones (90%) from WT mice produced IL-10, and a much smaller proportion (37%) released this cytokine from IFN-–/– mice (Fig. 2C). If V8.1/8.2+ cells were analyzed, the disparity in proportions was similar to that found for all clones (Fig. 2D). These data demonstrate that the absence of cytokines skewed not only the TCR profile but also the generation of critically important cytokines by T cells.
Reactivity to F3. The protective domain of rhsp 60 spans amino acids 172 to 443 (7). Immunization with polypeptide induces a protective immune response in mice. Previously, we demonstrated that among V8.1/8.2+ monoclonal T cells from C57BL/6 mice, recognition of this polypeptide was another determinant that distinguished a protective T cell from a nonprotective one (27). Hence, we assessed reactivity among our clones to it. The proportion of all clones recognizing F3 was markedly diminished (P < 0.01) among those derived from IFN-–/– mice and less so (P < 0.05) from IL-10–/– mice (Fig. 2E). A similar profile was true if only V8.1/8.2+ cells were analyzed (Fig. 2F).
Is the alteration in TCR usage a result of defective antigen presentation To examine whether deficient animals demonstrated altered antigen presentation, splenocytes from wild-type and cytokine-deficient animals were harvested and irradiated for use as antigen-presenting cells. Irradiated splenocytes from IFN-–/– mice manifested an ability to stimulate a V8.1/8.2+ clone similar to that of antigen-presenting cells from WT mice (Fig. 3). Data for one concentration of rhsp 60, 5 μg/ml, are shown. Similar findings were noted with 10 and 1 μg/ml (data not shown).
Protection-mediated rhsp 60 at the single-cell level is dependent on the coexpression of IFN- and IL-10. WT, IFN-–/–, and IL-10–/– mice were twice immunized at an interval of 2 weeks with rhsp 60. Splenocytes from immunized mice were transferred into TCR /–/– mice that were infected with 5 x 105 yeasts and monitored for survival. Mice receiving cells from WT animals manifested a prolonged survival compared to that of each of the other groups (Fig. 4A). Cultures of lungs and spleens at the termination of the experiment revealed that the organs of the surviving mice contained no detectable H. capsulatum CFU.
We previously demonstrated that the protective V8.1/8.2+ T cells must be reactive to F3 and produce IFN- (27). The clone that we utilized for those experiments also produced IL-10 (26). Thus, we sought to extend those studies and determine if coexpression of IL-10 and IFN- in conjunction with F3 reactivity was necessary to confer protection. To accomplish this, we utilized a V8.1/8.2+ T-cell clone that reacted to F3 that was derived from IL-10–/– mice (clone 6b) and clone D, which was the original clone derived from WT mice (26). In addition, another V8.1/8.2+ T-cell clone (clone 6) that coexpressed IL-10 and IFN- and reacted to F3 was used. T cells were transferred into TCR /–/– mice, and their survival was monitored. As seen in Fig. 4B, only clones that secreted both IL-10 and IFN- significantly (P < 0.01) prolonged survival.
DISCUSSION
rhsp 60 is a protective antigen that stimulates an increased production of IFN-, IL-10, and IL-12 by mouse splenocytes and causes the expansion of murine T cells that predominantly express V8.1/8.2 (8, 27). More importantly, subsets of V8.1/8.2+ T cells that are F3 reactive and produce IFN- are able to confer a protective response to T-cell-deficient mice (27). Thus, defined populations of T cells are involved in the generation of the protective response to rhsp 60. In results presented herein, we explored the possibility that the absence of IFN- or IL-10 would modulate the T-cell response to rhsp 60. We demonstrated that, indeed, the a lack of either one altered the frequency of T cells expressing V8.1/8.2, the cytokine profile of these T cells, and the proportion that reacted to the protective domain of this antigen. The results were more profound in mice that lacked IFN-. These findings indicate that the presence or absence of a cytokine necessary for protection has effects beyond the mere absence of that cytokine. The repertoire of T cells which the host utilizes to combat infection is in part shaped by the cytokine environment.
Among the alterations associated with the absence of IFN- was the marked decrement in the proportion of V8.1/8.2+ cells that are key in the generation of vaccine-induced immunity (27). One explanation for the altered TCR repertoire generated in the absence of IFN- is a defect in antigen presentation. IFN- regulates a number of effects on antigen presentation. These effects include increases in class I and class II major histocompatibility complex antigens, induction of lysosomal cathepsins that are important in the generation of immunogenic peptides, and induction of the proteosome pathway (4). This explanation appears to be unlikely since antigen-presenting cells from the IFN-–/– mice were as efficient as those from WT mice in stimulating T-cell responses. Alternatively, the altered TCR may have been a result of fewer V8.1/8.2+ cells in the spleens of animals, but this was not the case. The failure of this T-cell population to expand following vaccination in IFN-–/– as well as IL-10–/– mice remains unknown. The results unequivocally demonstrate that the presence or absence of cytokines may shape the TCR repertoire to an antigen or antigens.
Cytokine production among all T-cell clones was altered compared to that in cells from WT animals. In IL-10–/– mice, the proportion of Th1 cells was nearly 100% of that in WT animals. The absence of IL-10 greatly influenced the generation of Th1 cells, as has been observed previously. In mice, the absence of IL-10 or the blockade of its cognate receptor enhances the protective immune response to Leishmania donovani, Plasmodium spp., Listeria monocytogenes, Coccidioides immitis, Mycobacterium spp., and H. capsulatum (10, 13, 20, 23, 24, 28). Despite the pronounced skewing of the immune response to a Th1 phenotype, neither polyclonal nor monoclonal cells from IL-10–/– mice induced protection in TCR /–/– mice.
Additional analysis revealed that the proportion of T-cell clones that reacted to the protective domain of rhsp 60, F3, was decreased compared to that in WT mice whether all clones were examined or whether the examination was restricted to V8.1/8.2+ cells. Since responsiveness to this polypeptide is an important functional feature of protective T cells, the results indicate that IFN- and IL-10 are important for the development of this population. The failure of clone 6b, which did not produce IL-10, to confer protection may be a result the absence of this cytokine in the antifungal effector pathway. Other explanations should be considered. It is possible that clones that do not produce IL-10 are unable to traffic in a manner similar to that of clones that do release IL-10. In addition, the in vivo survival of clones that lack IL-10 may be impaired compared to that of IL-10+ clones. Nevertheless, our results demonstrate that the cytokine environment has a major impact on the type of T cells that emerge following immunization with a single protein.
The importance of IFN- and IL-10 in response to a pathogen has been documented in several models of infection. In most cases, the production of IL-10 or IFN- serves to inhibit the production of the other. This balance is vital in maintaining the integrity of the immune response without generating immunopathology at the site of infection. In our vaccination model, however, the presence of IL-10 may be necessary to obviate a massive inflammatory response associated with Th1 cells and to confer protection that is unrelated by upregulating antifungal effectors (22). The requirement for IL-10 in the generation of a protective immune response is uncommon. This cytokine is essential for mice infected with L. major to maintain protective immunity, principally because its presence allows low numbers of parasites to persist. Their persistence sustains the protective immune response (3). In addition, IL-10 has been reported to be important for the enhanced protection associated with pneumococcal polysaccharide vaccine and for several tumor vaccines (14, 19, 21).
The importance of endogenous IL-10 for the optimal expression of immunity mediated by rhsp 60 is linked to the production of IFN-. Only dual-expression clones that also reacted to F3 were protective. The mechanisms by which these two cytokines interact to trigger a protective response by a population of T cells are unclear. Production of IL-10 has been shown to limit the apoptosis of antigen-presenting cells and thus extend the time of antigen processing and presentation (5). Delivery of this cytokine by T cells may enhance the efficiency of antigen processing and prolong the time in which antigens may be recognized. Moreover, it is possible that the production of IL-10 by T cells may protect them from apoptosis and thus extend their functional life spans. Accordingly, the ability of IFN-+ IL-10+ T cells to promote immunity may exceed that of IFN-+ IL-10– cells. Work is under way to test this hypothesis.
We previously reported that the major source of IL-10 in splenocytes from vaccinated mice was not of T-cell origin (8). However, it is evident that T cells are a source—if not the major source. The findings suggest that analysis of polyclonal populations is not sufficiently sensitive to locate critically important T-cell populations that function to protect.
In survival studies, the transfer of cells from WT mice immunized with rhsp 60 conferred sterilizing immunity, whereas the transfer of a T-cell clone even though it was a Th1 clone did not induce the same type of protection. Although mice that received a protective clone exhibited prolonged survival, sterilizing immunity was not achieved. The most likely explanation for this finding is that protective immunity relies upon the interaction with other T-cell families. Thus, a clonal population may not be sufficient in a mouse that lacks all T cells that bear the / receptor.
In summary, we have demonstrated that the absence of IFN- and, to a lesser extent, IL-10 influences a number of critical markers of protective T cells that are generated in response to vaccination with rhsp 60. These findings have implications for T-cell vaccines that rely predominantly if not exclusively on T cells for combating the infectious process.
ACKNOWLEDGMENTS
This work was supported by grants AI34361 and AI42747 from the National Institutes of Health and by a Merit Review grant from the Department of Veterans Affairs.
We thank Reta Gibbons for technical assistance.
Present address: Tupper Research Institute, Division of Geographic Medicine and Infectious Diseases, New England Medical Center, Tufts University School of Medicine, Boston, MA 02111.
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