Immunol. Cell Biol. (1990)68, 179-185
Murine candidiasis: Susceptibility is associated with the induction of T cell-mediated, strain-specific autoreactivity R. B. Ashman Department of Pathology, University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, WA 6009, Australia (Submitted 28 February 1990. Accepted for publication 30 April 1990.) Summary Inbred mice can be classified as susceptible or resistant to systemic infection with the yeast Candida albicans by histopathological evaluation of tissue lesions. Candida-specific memory T cell responses generated by resistant BALB/c mice are vigorous and sustained, whereas those displayed by susceptible CBA/H mice are weak. When spleen cells from immune mice were activated by culture with Candida antigens in vitro, and injected into syngeneic and allogeneic recipients in the absence of further antigenic stimulation, cells from CBA/H mice induced a specific inflammatory response only in CBA/H recipients. In contrast, cells from immune BALB/c mice showed no specific activity. The effector cells were identified as T cells of the cytotoxic/suppressor subclass (CD4-, CD8+); and analysis in various Fl hybrid mice showed that reactivity was expressed only in animals carrying CBA/H genes. The data thus indicate that susceptibility to C albicans infection is associated with the induction of a T cell subpopulation that has the potential to react specifically against unmodified self antigens expressed by the susceptible strain.
INTRODUCTION Susceptibility or resistance to infectious agents including viruses, bacteria and parasites, is known to be governed by genes mapping both inside, and outside, the major histocompatibility complex (MHC). T cell-mediated immune responses are controlled directly by MHC genes, and in diseases such as lymphocytic choriomeningitis virus infection, the severity of the disease shows a strong correlation with the magnitude of cell-mediated immune responses (1,2). In other infections in which the immunopathological component is not as strong, the severity of tissue lesions appears to be determined by innate resistance mechanisms that are controlled by non-MHC genes (3). Infections caused by the yeast Candida albicans, a common opportunistic pathogen, are subject similarly to complex genetic regulation. Immune/inflammatory responses in the popliteal lymph nodes during a primary infection in mice are regulated by MHC genes (4,5); however, the histological severity of organ lesions is determined by genes mapping outside the MHC (6). The prototypes of resistant and susceptible strains are BALB/c mice, that show Abbreviations used in this paper: IL-3, interleukin-3; MHC, major histocompatibility complex.
high and long-lasting inflammatory responses, but mild tissue lesions, and CBA/H mice, that exhibit weak inflammatory responses and severe tissue lesions. An important observation in this experimental system is that memory T cell responses, such as delayed-type hypersensitivity and Candida-specific lymphocyte proliferation, show a direct correlation with disease susceptibility (7). In contrast to the primary infection, the magnitude of memory T cell responses appears to be governed by non-MHC genes. This report analyses further the relationships between susceptibility and the cell-mediated immune response, and shows that susceptibility is associated with the production, in CBA/H but not in BALB/c mice, of T cells that have the potential to react against unmodified self antigens. MATERIALS AND METHODS Mice Specific pathogen-free mice, 8-12 weeks of age, were purchased from the Animal Resources Centre, Perth. The animals were housed and used in accordance with the National Health and Medical Research Council's 'Code of Practice for the Care and Use of Animals for Experimental Purposes in Australia'. The mice are subjected to routine microbiological screening, and do not carry C. albicans in the gut. Only female mice were used in experiments.
180
R. B. ASHMAN
Yeast Candida albicans isolate 3630 was obtained from the Mycology Reference Laboratory at the Royal North Shore Hospital, Sydney, and maintained by serial passage on Sabouraud's agar slopes. For use, yeasts were grown for 18 h in Sabouraud's broth at'iO'Cwith continuous agitation, washed three times in normal saline and adjusted to the appropriate concentration for inoculation into the mice. Immunization BALB/c and CBA/H mice were injected intravenously with 1 X 105 Candida albicans blastospores, prepared as described above. Viable organisms could be recovered from the spleens of immunized mice for approximately three weeks, so the animals were routinely rested for 6-8 weeks before use. Antigen preparation A Candida antigen was prepared as follows: organisms were grown at 30°C in 5 L of Sabouraud's broth for 2 months with constant stirring. Before use, the culture was tested for purity by the State Health Laboratories, Queen Elizabeth II Medical Centre. The yeasts were recovered by centrifugation and washed three times in normal saline. The packed cells were weighed, resuspended to 20 mL and disrupted for 4 min in a Braun MSK Homogenizer (Braun Instrument Company, 805 Grandview Drive South, San Francisco, California, USA) using 0.45-0.5 mm glass beads. The cellular debris was removed by centrifugation. The supernatant was tested for sterility by culture on Sabouraud's agar plates, and stored in small aliquots at — 20°C. Optimum concentrations for stimulation of lymphocyte proliferation were determined empirically. Cell culture Immune spleen cells were activated by culture in vitro with antigen-treated syngeneic spleen cells. In brief, spleens were removed aseptically, minced, and lymphocytes recovered by centrifugation over Isopaque/Ficoll graients. The normal cells were treated for 30 min at 37°C with a 1:10 dilution of Candida antigen in 1 mL RPMl 1640 medium supplemented with 5% fetal calf serum and lO—* mol/L 2 mercaptoethanol, washed once, and then prevented from replication by treatment with Mitomycin C. Cultures consisting of 8 X lO'' immune cells and 2 X 1 0 ' antigen-treated normal cells in 40 mL of medium were incubated for 5 days at 37°C in 5% CO2. The activated cells were then harvested, centrifuged over Isopaque/ Ficoll gradients, washed and adjusted to the appropriate concentrations for injection into the mice. Fluorescence analysis Cells were activated by in vitro culture as described above and stained with monoclonal antibodies against cell-surface antigens using a standard protocol for indirect immunofluorescence (8). The antibodies used were: clone 30H12, specific for Thy 1 2; clone GKl 5,
specific for L3T4; and clone 53 6 72, specific for Lyt 2. The secondary antibody was a fluoresceinated sheep anti-rat antiserum (Silenius Laboratories, Hawthorn, Victoria). Fluorescent cells were quantitated using a Becton-Dickinson FACS Analyser 1. Footpad swelling reaction Cultured cells were adjusted to 2X10'/mL and 50 nL (1 X 106 cells) injected into the footpad. Swelling was measured after 24 h using Mitutoyo dial calipers. Depletion of lymphocyte subsets Cultured cells were treated with the following monoclonal antibodies: clone 6 68, specific for Thy 1 -2, at a final concentration of 1:1000; 31 mol/L, specific for L3T4, at 1:10; and clone 172, specific for Lyt 2, at 1:10. The cells were incubated with the antisera at 4°C for 30 min, washed once, and then incubated with a 1:10 dilution of low toxicity rabbit complement for 30 min at 37°C. After final washing, the cells were diluted to the original volume, and the equivalent of 10* cells in 50 (xL injected into the footpad. Cytotoxicity assay Thioglycollate-induced macrophage target cells, and the natural-killer sensitive YAC-1 cells were labelled with "Cr, and the Candida activated T cells tested for their cytotoxic potential in a 6 h assay. Lymphokine assays Activated T cells were harvested from 5 day cultures, washed twice and challenged with either syngeneic or allogeneic antigen-treated cells, or untreated control cells, for 6 h. The supernatants were harvested, and tested for their ability to support the growth of FD cells (an IL-3 (interleukin-3) dependent cell line). Recombinant IL-3 (a gift from Dr A. Hapel) was used as a control. Proliferation was measured by reduction of a tetrazolium dye (9). Statistics Experiments were repeated at least three times. Comparisons were made using Student's Mest and the paired Mest.
RESULTS Reactivity of activated spleen cells Spleen cells from BALB/c and CBA/H mice that have recovered from an initial infection with C. albicans respond by specific proliferation to in vitro stimulation with Candida antigen (7). Preliminary experiments showed that activated cells from these cultures produced inflammation and swelling after injection into the footpad of syngeneic mice. In order to evaluate the specificity of this reaction, activated cells from both BALB/c and CBA/H mice were injected into the
AUTOREACTIVE T CELLS IN MOUSE CANDIDIASIS
right and left footpad respectively of both BALB/c atid CBA/H mice. Injection ofthe antigen-stimulated BALB/c cells controlled for the effects of non-specific inflammation in CBA/H mice, and vice versa. The actual meati thickness of the footpads in each strain 24 h after injection, as well as the mean diflerence between left and right footpads, is shown in Table 1. CBA/H mice showed a specific response to the activated CBA/H cells, whereas swelling in the BALB/c mice was non-specific. Strain-specificity of antigen recognition In order to determine whether these T cell responses were directed against antigens in the MHC, activated cells were assayed in CBA/H and BALB/c recipients, in [BALB/c X BALB/cH-2k] Fl hybrid mice that carry both CBA/H and BALB/c MHC haplotypes, and in [BALB/c X CBA/H] Fl hybrid mice. Reactivity was observed only in mice that expressed CBA/H antigens (Table 2), and it should be noted that footpad swelling in [BALB/c and CBA/H] Fl hybrid mice was comparable to that in CBA/H controls, indicating that the presence of BALB/c background genes did not influence the magnitude of the inflammatory response. The failure of activated CBA-H cells to induce reactivity in [BALB/c X BALB/c-H-2'
Murine candidiasis: Susceptibility is associated with the induction of T cell-mediated, strain-specific autoreactivity R. B. Ashman Department of Pathology, University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, WA 6009, Australia (Submitted 28 February 1990. Accepted for publication 30 April 1990.) Summary Inbred mice can be classified as susceptible or resistant to systemic infection with the yeast Candida albicans by histopathological evaluation of tissue lesions. Candida-specific memory T cell responses generated by resistant BALB/c mice are vigorous and sustained, whereas those displayed by susceptible CBA/H mice are weak. When spleen cells from immune mice were activated by culture with Candida antigens in vitro, and injected into syngeneic and allogeneic recipients in the absence of further antigenic stimulation, cells from CBA/H mice induced a specific inflammatory response only in CBA/H recipients. In contrast, cells from immune BALB/c mice showed no specific activity. The effector cells were identified as T cells of the cytotoxic/suppressor subclass (CD4-, CD8+); and analysis in various Fl hybrid mice showed that reactivity was expressed only in animals carrying CBA/H genes. The data thus indicate that susceptibility to C albicans infection is associated with the induction of a T cell subpopulation that has the potential to react specifically against unmodified self antigens expressed by the susceptible strain.
INTRODUCTION Susceptibility or resistance to infectious agents including viruses, bacteria and parasites, is known to be governed by genes mapping both inside, and outside, the major histocompatibility complex (MHC). T cell-mediated immune responses are controlled directly by MHC genes, and in diseases such as lymphocytic choriomeningitis virus infection, the severity of the disease shows a strong correlation with the magnitude of cell-mediated immune responses (1,2). In other infections in which the immunopathological component is not as strong, the severity of tissue lesions appears to be determined by innate resistance mechanisms that are controlled by non-MHC genes (3). Infections caused by the yeast Candida albicans, a common opportunistic pathogen, are subject similarly to complex genetic regulation. Immune/inflammatory responses in the popliteal lymph nodes during a primary infection in mice are regulated by MHC genes (4,5); however, the histological severity of organ lesions is determined by genes mapping outside the MHC (6). The prototypes of resistant and susceptible strains are BALB/c mice, that show Abbreviations used in this paper: IL-3, interleukin-3; MHC, major histocompatibility complex.
high and long-lasting inflammatory responses, but mild tissue lesions, and CBA/H mice, that exhibit weak inflammatory responses and severe tissue lesions. An important observation in this experimental system is that memory T cell responses, such as delayed-type hypersensitivity and Candida-specific lymphocyte proliferation, show a direct correlation with disease susceptibility (7). In contrast to the primary infection, the magnitude of memory T cell responses appears to be governed by non-MHC genes. This report analyses further the relationships between susceptibility and the cell-mediated immune response, and shows that susceptibility is associated with the production, in CBA/H but not in BALB/c mice, of T cells that have the potential to react against unmodified self antigens. MATERIALS AND METHODS Mice Specific pathogen-free mice, 8-12 weeks of age, were purchased from the Animal Resources Centre, Perth. The animals were housed and used in accordance with the National Health and Medical Research Council's 'Code of Practice for the Care and Use of Animals for Experimental Purposes in Australia'. The mice are subjected to routine microbiological screening, and do not carry C. albicans in the gut. Only female mice were used in experiments.
180
R. B. ASHMAN
Yeast Candida albicans isolate 3630 was obtained from the Mycology Reference Laboratory at the Royal North Shore Hospital, Sydney, and maintained by serial passage on Sabouraud's agar slopes. For use, yeasts were grown for 18 h in Sabouraud's broth at'iO'Cwith continuous agitation, washed three times in normal saline and adjusted to the appropriate concentration for inoculation into the mice. Immunization BALB/c and CBA/H mice were injected intravenously with 1 X 105 Candida albicans blastospores, prepared as described above. Viable organisms could be recovered from the spleens of immunized mice for approximately three weeks, so the animals were routinely rested for 6-8 weeks before use. Antigen preparation A Candida antigen was prepared as follows: organisms were grown at 30°C in 5 L of Sabouraud's broth for 2 months with constant stirring. Before use, the culture was tested for purity by the State Health Laboratories, Queen Elizabeth II Medical Centre. The yeasts were recovered by centrifugation and washed three times in normal saline. The packed cells were weighed, resuspended to 20 mL and disrupted for 4 min in a Braun MSK Homogenizer (Braun Instrument Company, 805 Grandview Drive South, San Francisco, California, USA) using 0.45-0.5 mm glass beads. The cellular debris was removed by centrifugation. The supernatant was tested for sterility by culture on Sabouraud's agar plates, and stored in small aliquots at — 20°C. Optimum concentrations for stimulation of lymphocyte proliferation were determined empirically. Cell culture Immune spleen cells were activated by culture in vitro with antigen-treated syngeneic spleen cells. In brief, spleens were removed aseptically, minced, and lymphocytes recovered by centrifugation over Isopaque/Ficoll graients. The normal cells were treated for 30 min at 37°C with a 1:10 dilution of Candida antigen in 1 mL RPMl 1640 medium supplemented with 5% fetal calf serum and lO—* mol/L 2 mercaptoethanol, washed once, and then prevented from replication by treatment with Mitomycin C. Cultures consisting of 8 X lO'' immune cells and 2 X 1 0 ' antigen-treated normal cells in 40 mL of medium were incubated for 5 days at 37°C in 5% CO2. The activated cells were then harvested, centrifuged over Isopaque/ Ficoll gradients, washed and adjusted to the appropriate concentrations for injection into the mice. Fluorescence analysis Cells were activated by in vitro culture as described above and stained with monoclonal antibodies against cell-surface antigens using a standard protocol for indirect immunofluorescence (8). The antibodies used were: clone 30H12, specific for Thy 1 2; clone GKl 5,
specific for L3T4; and clone 53 6 72, specific for Lyt 2. The secondary antibody was a fluoresceinated sheep anti-rat antiserum (Silenius Laboratories, Hawthorn, Victoria). Fluorescent cells were quantitated using a Becton-Dickinson FACS Analyser 1. Footpad swelling reaction Cultured cells were adjusted to 2X10'/mL and 50 nL (1 X 106 cells) injected into the footpad. Swelling was measured after 24 h using Mitutoyo dial calipers. Depletion of lymphocyte subsets Cultured cells were treated with the following monoclonal antibodies: clone 6 68, specific for Thy 1 -2, at a final concentration of 1:1000; 31 mol/L, specific for L3T4, at 1:10; and clone 172, specific for Lyt 2, at 1:10. The cells were incubated with the antisera at 4°C for 30 min, washed once, and then incubated with a 1:10 dilution of low toxicity rabbit complement for 30 min at 37°C. After final washing, the cells were diluted to the original volume, and the equivalent of 10* cells in 50 (xL injected into the footpad. Cytotoxicity assay Thioglycollate-induced macrophage target cells, and the natural-killer sensitive YAC-1 cells were labelled with "Cr, and the Candida activated T cells tested for their cytotoxic potential in a 6 h assay. Lymphokine assays Activated T cells were harvested from 5 day cultures, washed twice and challenged with either syngeneic or allogeneic antigen-treated cells, or untreated control cells, for 6 h. The supernatants were harvested, and tested for their ability to support the growth of FD cells (an IL-3 (interleukin-3) dependent cell line). Recombinant IL-3 (a gift from Dr A. Hapel) was used as a control. Proliferation was measured by reduction of a tetrazolium dye (9). Statistics Experiments were repeated at least three times. Comparisons were made using Student's Mest and the paired Mest.
RESULTS Reactivity of activated spleen cells Spleen cells from BALB/c and CBA/H mice that have recovered from an initial infection with C. albicans respond by specific proliferation to in vitro stimulation with Candida antigen (7). Preliminary experiments showed that activated cells from these cultures produced inflammation and swelling after injection into the footpad of syngeneic mice. In order to evaluate the specificity of this reaction, activated cells from both BALB/c and CBA/H mice were injected into the
AUTOREACTIVE T CELLS IN MOUSE CANDIDIASIS
right and left footpad respectively of both BALB/c atid CBA/H mice. Injection ofthe antigen-stimulated BALB/c cells controlled for the effects of non-specific inflammation in CBA/H mice, and vice versa. The actual meati thickness of the footpads in each strain 24 h after injection, as well as the mean diflerence between left and right footpads, is shown in Table 1. CBA/H mice showed a specific response to the activated CBA/H cells, whereas swelling in the BALB/c mice was non-specific. Strain-specificity of antigen recognition In order to determine whether these T cell responses were directed against antigens in the MHC, activated cells were assayed in CBA/H and BALB/c recipients, in [BALB/c X BALB/cH-2k] Fl hybrid mice that carry both CBA/H and BALB/c MHC haplotypes, and in [BALB/c X CBA/H] Fl hybrid mice. Reactivity was observed only in mice that expressed CBA/H antigens (Table 2), and it should be noted that footpad swelling in [BALB/c and CBA/H] Fl hybrid mice was comparable to that in CBA/H controls, indicating that the presence of BALB/c background genes did not influence the magnitude of the inflammatory response. The failure of activated CBA-H cells to induce reactivity in [BALB/c X BALB/c-H-2'
