Recognition of Histoplasma capsulatum yeast-cell antigens by human lymphocytes and human T-cell clones.

We have prepared a detergent extract from the cell wall and cell membrane of Histoplasma capsulatum yeast cells that is recognized by T cells from mice immunized with viable organisms or with the extract. A 62-kd antigen from this extract has been isolated and has been shown to be antigenic and to confer protective immunity in mice. In this study, we examined the in vitro proliferative response by human lymphocytes and human T-cell clones to both the extract from the cell wall and cell membrane and the 62-kd antigen, termed HIS-62. Seven healthy individuals were identified whose peripheral blood mononuclear cells responded to the cell wall and cell membrane extract from H. capsulatum yeast cells. Mononuclear cells from all seven individuals recognized histoplasmin. T-cell clones were generated from peripheral blood mononuclear cells from a single person using the extract from cell wall and cell membrane as the antigenic preparation. Nineteen clones were propagated; all expressed the surface phenotype CD3+, CD4+, T-cell receptor alpha/beta +. The clones were antigen-specific. Of 17 clones studied, none responded to extracts from Blastomyces dermatitidis or Coccidioides immitis or to tetanus toxoid. Sixteen of 19 clones recognized HIS-62 in vitro. Additional analysis revealed that cells from each of the seven individuals who responded to the extract mounted a proliferative response to HIS-62. Thus, the extract from the cell wall and cell membrane and HIS-62 are targets of the human cell-mediated immune response to H. capsulatum. Moreover, HIS-62 appears to be an immunodominant antigen.     

Antigenicity and immunogenicity of an extract from the cell wall and cell membrane of Histoplasma capsulatum yeast cells.

In order to identify T-cell antigens from Histoplasma capsulatum yeast cells, we prepared a detergent extract of the cell wall and cell membrane of yeast-phase H. capsulatum G217B and analyzed its antigenicity and immunogenicity. Mice injected with viable H. capsulatum yeast cells or with 500 or 1,000 micrograms of the extract mounted a delayed-type hypersensitivity response to solubilized cell wall and cell membrane. Vaccination with this antigenic preparation conferred a protective immune response in mice that were challenged intravenously with H. capsulatum yeast cells. The extract induced in vitro proliferation by splenocytes from mice injected with either viable yeast cells or the soluble cell wall and cell membrane preparation. We also examined the profile of in vitro responses by a murine T-cell line and by cloned T cells to soluble cell wall and cell membrane by employing the technique of T-cell immunoblotting. Two prominent regions that stimulated the T-cell line and cloned T cells were identified. Fractions encompassing an area between 53 and 64 kDa caused proliferation by a T-cell line and five of six clones. Antigens recognized by the T-cell line and by three of six clones were contained in another area that extended from 69 to 82 kDa. The data demonstrate that this soluble extract from cell wall and cell membrane contains antigens recognized by T cells and mediates protective immunity. Moreover, T-cell immunoblotting provides a useful technique for mapping immunoreactive molecules from H. capsulatum yeast cells.     

An 80-kilodalton antigen from Histoplasma capsulatum that has homology to heat shock protein 70 induces cell-mediated immune responses and protection in mice.

An extract of the cell wall and cell membrane from Histoplasma capsulatum yeast cells was assayed by Western blot (immunoblot) for reactivity with two monoclonal antibodies to heat shock protein 70. Four bands with molecular masses of 80, 66, 54, and 32 kDa bound both antibodies. The 80-kDa protein was isolated, analyzed for homology to heat shock protein 70, and tested for antigenicity and immunogenicity in C57BL/6 mice. The 80-kDa protein reacted with monoclonal antibody to heat shock protein 70. Sera from mice immunized with the antigen recognized H. capsulatum heat shock protein 70. Moreover, the amino-terminal sequence of the 80-kDa protein revealed substantial homology with heat shock protein 70 from several species. The 80-kDa protein induced delayed-type hypersensitivity responses in mice immunized with either viable yeast cells or antigen. Splenocytes from mice immunized with yeast cells or with antigen responded in vitro to the 80-kDa antigen. Immunization of mice with the antigen enhanced host resistance against a sublethal inoculum of H. capsulatum yeast cells, but it did not reduce the mortality of mice given a lethal challenge of yeast cells. Thus, this antigen manifests homology with members of the heat shock protein 70 family. Furthermore, the 80-kDa protein elicits cellular immune responses to H. capsulatum, and it mediates protective immunity.     

Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones.

Experimental studies have suggested that antigen-specific T lymphocytes are important mediators of resistance to infection with the pathogenic fungus Histoplasma capsulation. To gain a better understanding of the role of T lymphocytes, we developed murine T-cell lines and clones that recognized Histoplasma antigens. These T cells were of the helper/inducer phenotype (Thy-1.2+ Lyt-1+ L3T4+ Lyt-2-) and exerted multiple immunological functions. T-cell lines and 12 clones proliferated vigorously in response to histoplasmin; the T-cell lines and 6 clones also were reactive with heterologous fungal antigens prepared from either Blastomyces dermatitidis or Coccidioides immitis. Recognition of antigen by T cells was H-2 restricted; in the absence of antigen, four clones demonstrated alloreactivity. All T-cell clones conferred local delayed-type hypersensitivity responses when injected with antigen into footpads of mice. Ten of 12 T-cell clones released interleukin-2 after stimulation with antigen, and all clones tested secreted interferon. Moreover, culture supernatants from antigen-stimulated clones armed peritoneal macrophages to inhibit intracellular growth of H. capsulatum yeast cells. All clones assayed exerted nonspecific help. Thus, development of T-cell clones should facilitate analysis of the regulatory properties of Histoplasma-specific T cells.     

A novel 62-kilodalton egg antigen from Schistosoma mansoni induces a potent CD4(+) T helper cell response in the C57BL/6 mouse

In infection with Schistosoma mansoni, hepatic granuloma formation is mediated by CD4(+) T helper (Th) cells sensitized to schistosomal egg antigens. There is considerable variation among infected individuals with respect to both severity of disease and the T-cell response to egg antigens. In the BL/6 mouse, the egg granulomas are relatively small and the relevant sensitizing egg antigens are largely unknown. We investigated the CD4(+) Th cell response of infected BL/6 mice to egg antigens fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found a prominent lymphoproliferative response to be directed against a 62-kDa component. With the aid of a specific T-cell hybridoma, 4E6, the 62-kDa antigen was isolated; following partial digestion with endoproteinase Glu-C, an internal amino acid sequence was found to be identical with one present in the enzyme phosphoenolpyruvate carboxykinase (PEPCK) of the organisms Caenorhabditis elegans and Treponema pallidum and to differ by one residue from PEPCK of various other species. In CD4(+) Th cells from 7.5- 8.5-week-infected BL/6 mice, the purified 62-kDa molecule elicited a potent proliferative response which, based on cytokine analysis, was of a mixed Th-1 and Th-2 type. Our results reveal a novel egg antigen of particular prominence in the BL/6 mouse and suggest that the immune response in schistosomiasis is a product of sensitization to egg antigens that may vary considerably in immunogenicity from strain to strain.     

Differential antigen recognition by T cell populations from strains of mice developing polar forms of granulomatous inflammation in response to eggs of Schistosoma mansoni

In humans, infection with schistosome helminths can lead to dissimilar forms of clinical disease. Likewise, in the experimental mouse system, identical infection protocols with Schistosoma mansoni cause a more severe granulomatous disease in the C3H strain than in the C57BL/6 strain. To address this difference, we developed panels of schistosomal egg antigen (SEA)-specific T cell hybridomas to compare the responses of C3H and C57BL/6 mice to the major egg antigen p40. All derived C3H T cell hybridomas, despite being clonally distinct and restricted by either I-A^k or I-E^k, responded to recombinant fragment 15-1 of the p40 antigen, while none of the C57BL/6 T cell hybridomas did. Consistent with the observed monoclonal T cell responses, polyclonal lymph node cells from schistosome-infected C3H mice reacted strongly to fragment 15-1, which contrasted sharply with the weak response displayed by the C57BL/6 strain. Moreover, studies with congenic mice demonstrated that the strong CD4⁺ T cell response to fragment 15-1 was under major histocompatibility complex control and segregated with the H-2^k haplotype. These findings suggest that a dominant T cell response against a major egg antigen may represent a risk factor for the development of severe disease.     

Protective immunity in murine histoplasmosis: functional comparison of adoptively transferred T-cell clones and splenic T cells.

In this study, I examined whether a murine T-cell line and three clones that recognize Histoplasma capsulatum antigens in vitro could confer protection in vivo against a challenge of Histoplasma yeasts. C57BL/6 mice were each inoculated with 5 X 10(4) yeasts intravenously; 1 h later, 5 X 10(6) or 2 X 10(7) resting T cells were inoculated intravenously. At week 1 of infection, the T-cell line and all clones failed to reduce the number of H. capsulatum CFU in the spleens of mice compared with numbers in infected controls. Administration of recombinant interleukin 2 or cyclophosphamide to infected mice did not potentiate the functional activity in vivo of either the T-cell line or the clones. In contrast, inoculation with 2 X 10(7) CD4+ but not CD8+ cells isolated from the spleens of mice immunized with 10(6) viable yeast cells sharply diminished the number of CFU in the spleens of infected animals. Moreover, splenic CD4+ cells from immune mice transferred a delayed-type hypersensitivity response, whereas the T-cell line and clones did not. Injection Injection of an equal number of cloned T cells and CD8+ splenocytes from immune mice did not transfer resistance to infected mice. Additional studies were undertaken to determine if the ineffectiveness of cloned T cells was associated with a failure to migrate to and survive within spleens of infected mice. B6.PL Thy-1a/Cy mice, which are genetically identical to C57BL/6 mice except that T cells of the former bear Thy-1.1 rather than Thy-1.2, were inoculated with Histoplasma yeasts and then injected with immune CD4+ splenocytes or a T-cell clone. At days 1 and 7 of infection, virtually no Thy-1.2+ cells were detected in the spleens of infected mice given cloned T cells. However, the spleens of animals inoculated with immune CD4+ cells contained a small but significant (P less than 0.01) proportion of Thy-1.2+ cells at both day 1 and day 7 postinoculation of H. capsulatum. Thus, the failure of T-cell clones to transfer protection against H. capsulatum may be explained by defective trafficking or poor survival in vivo or both.     

Functional analysis of Histoplasma capsulatum-reactive T-cell hybridomas.

Activation of CD4+ T cells is a crucial step in the elimination of Histoplasma capsulatum yeast cells from tissues. However, only a limited amount of information exists concerning the immunobiology of H. capsulatum-reactive T cells that are CD4+. To facilitate the analysis of the functional activities of this T-cell subpopulation, we developed a panel of 10 murine T-cell hybridomas from splenocytes of immune C57BL/6 mice. All hybridomas reacted with monoclonal anti-CD4+ antibody and released interleukin-2 after stimulation with histoplasmin. Within 3 weeks, the reactivity of hybridomas to histoplasmin declined dramatically, yet the cells responded vigorously to yeast-phase preparations that were enriched for cytosol, cell wall, or cell membrane. Of 10 hybridomas studied, only one recognized heterologous fungal antigens. Responsiveness to yeast-phase antigens was restricted by I-Ab. We mapped determinants in cytosol and cell wall or cell membrane by the technique of one-dimensional T-cell immunoblotting. The patterns of responses of hybridomas to cytosol were nearly uniform. All hybridomas responded to two immunodominant regions in cytosol with masses ranging from less than or equal to 18 to 26 kilodaltons (kDa) and 35 to 39 kDa. All hybridomas tested responded to determinants in the cell wall or cell membrane preparation with masses of 35 to 39 kDa. These hybridomas provide a useful tool for defining yeast-phase antigens that trigger T-cell activation.     

Schistosoma mansoni phosphoenolpyruvate carboxykinase, a novel egg antigen: immunological properties of the recombinant protein and identification of a T-cell epitope

In schistosomiasis mansoni, hepatic granulomatous inflammation surrounding parasite eggs is mediated by CD4(+) T helper (Th) cells sensitized to schistosomal egg antigens (SEA). We previously showed that a prominent lymphoproliferative response of CD4(+) Th cells from schistosome-infected C57BL/6 (BL/6) mice was directed against a 62-kDa component of SEA. A partial amino acid sequence of the 62-kDa component was found to be identical with one present in the enzyme phosphoenolpyruvate carboxykinase (PEPCK). Based on this sequence, a cDNA clone containing the entire coding region of PEPCK was identified, and the full recombinant Schistosoma mansoni PEPCK (rSm-PEPCK) of 626 amino acids was purified from a prokaryotic expression system. rSm-PEPCK strongly stimulated a specific T-cell hybridoma, 4E6, as well as CD4(+) Th cells from SEA-immunized BL/6 mice and from infected BL/6, CBA, and BALB/c mice. In the infected mice, rSm-PEPCK elicited significant gamma interferon production as well as, to a lesser extent, production of interleukin-2 and -5. In BL/6 and BALB/c mice, the CD4(+) Th cell response to rSm-PEPCK was greater than that directed against the egg antigen Sm-p40; conversely, CBA mice responded better to Sm-p40 than to Sm-PEPCK. A 12-amino-acid region (residues 398 to 409: DKSKDPKAHPNS) was demonstrated to contain a T-cell epitope; synthetic peptides containing this epitope significantly stimulated specific hybridoma 4E6 and polyclonal CD4(+) Th cells. The identification and characterization of immunogenic egg components will contribute to the understanding and possible control of T-cell-mediated schistosomal disease.     

In vivo and in vitro characterization of murine T-cell clones reactive to Mycobacterium tuberculosis.

Seventeen helper T-cell clones were derived by stimulating lymph node cells from sensitized C57BL/6 mice with Mycobacterium tuberculosis H37Ra, M. tuberculosis H37Rv, or purified protein derivative. Most clones cross-reacted with Mycobacterium bovis BCG, H37Ra, H37Rv, and purified protein derivative. However, four clones were able to differentiate H37Rv from H37Ra, or BCG from H37Ra and H37Rv. In addition, four other T-cell clones recognized recombinant antigens of 19 and 65 kilodaltons isolated from a genomic expression library of M. tuberculosis by using monoclonal antibodies. All clones were Ia restricted and had the Thy-1.2+ Lyt-1+ L3T4+ Lyt-2- phenotype. On stimulation with antigen, all of the clones tested secreted interleukin-2 and gamma interferon but not B-cell stimulatory factor 1. All of the clones tested induced an antigen-specific delayed-type hypersensitivity response upon local cell transfer, although the magnitude of this response differed markedly among clones.     

Experimental histoplasmosis in the beige mouse

Mice carrying the beige mutation (bg/bg) on a C57Bl/6 background were challenged with Histoplasma capsulatum. bg/bg mice had higher mortality and higher lung tissue fungal counts in their lungs than either bg/+ or C57Bl/6 mice challenged with equal inocula. Immunologic studies showed that bg/bg mice developed normal delayed-type hypersensitivity (DTH) reactions to histoplasmin, but had deficient NK cell cytotoxic activity against YAC-1 target cells. Studies of macrophage killing of H. capsulatum in vitro showed that T lymphocytes of either bg/+ or bg/bg mice were able to activate fungal killing by bg/+ but not by bg/bg macrophages. These studies, while not excluding a role for the NK cell, suggest that macrophage dysfunction may be critical in the greater susceptibility of the bg/bg mouse and, by extension, that macrophage function is of major importance in host defense against H. capsulatum.     

Host Defenses in Experimental Scrub Typhus: Delayed-Type Hypersensitivity Responses of Inbred Mice

Delayed-type hypersensitivity responses of inbred mice during the course of lethal and chronic infections with strains of Rickettsia tsutsugamushi were evaluated by using the influx of radiolabeled cells into antigen-injected ears. Congenic strains of C3H mice, which previously have been shown to be resistant (C3H/RV) or sensitive (C3H/HeDub) to lethal intraperitoneal infection with the Gilliam strain of rickettsiae, both expressed delayed-type hypersensitivity early in the course of infection (5 to 7 days). The sensitive C3H/HeDub mice, however, exhibited a marked decline in reactivity just before death. In contrast, reactivity of C3H/RV mice remained high through day 9 and declined slowly through day 15 after infection. Similar results were obtained when BALB/c mice were infected with either the Karp or the Gilliam strain of rickettsiae, which produce a lethal or nonlethal infection, respectively, in this strain of mice. Rechallenge of C3H/RV mice elicited a rapid increase in reactivity, suggesting a secondary memory response. To analyze delayed-type hypersensitivity during chronic infection, C3H/HeDub mice were immunized by subcutaneous infection with the Gilliam strain of R. tsutsugamushi, and both delayed-type hypersensitivity reactivity and resistance to intraperitoneal challenge were examined. Delayed-type hypersensitivity reactivity developed slowly and peaked at 21 days postimmunization, which correlated with resistance to intraperitoneal challenge. Delayed-type hypersensitivity reactivity declined thereafter, but resistance to intraperitoneal challenge remained through 28 days postimmunization. Delayed-type hypersensitivity reactivity increased after secondary challenge at 28 days, again suggesting antigen memory generated by primary immunization. Transfer of delayed-type hypersensitivity reactivity was accomplished by using immune thymus-derived splenic lymphocytes isolated with nylon-wool columns. Abrogation of the ability of immune spleen cells to transfer delayed-type hypersensitivity reactivity after treatment with anti-Thy 1.2 alloantiserum and complement further supported the view that delayed-type hypersensitivity responses to scrub typhus rickettsiae were mediated by thymus-derived lymphocytes.     

Host-Etiological Agent Interactions in Intranasally and Intraperitoneally Induced Cryptococcosis in Mice

Inbred CBA/J mice were used in developing a defined in vivo model for studying host-parasite relationships in cryptococcosis. Mice were infected either intranasally or intraperitoneally with 10³ viable Cryptococcus neoformans cells. At weekly intervals over a 92-day period, C. neoformans growth profiles in the lungs, spleens, livers, and brains of the infected animals were determined. In addition, humoral and delayed-type hypersensitivity responses and cryptococcal antigen levels were assayed in these mice. Intranasally infected mice developed strong delayed-type hypersensitivity reactions in response to cryptococcal culture filtrate (CneF) antigen, and there was good correlation between acquisition of delayed-type hypersensitivity and the reduction of C. neoformans cell numbers in infected tissues. In contrast, intraperitoneally infected mice displayed greater numbers of C. neoformans cells in tissues and had somewhat suppressed delayed-type hypersensitivity responses to CneF antigen. Anticryptococcal antibodies were not detected in intranasally or intraperitoneally infected mice, but cryptococcal polysaccharide antigen titers were relatively high in both groups. The transfer of sensitized spleen cells from intranasally infected mice to syngeneic naive recipient mice resulted in the transfer of delayed-type hypersensitivity responsiveness to cryptococcal antigen in the recipients. The intranasally induced infection in mice was similar to the naturally acquired infection in humans; therefore we are proposing that this murine-cryptococcosis model would be useful in gaining a greater understanding of host-etiological agent relationships in this disease.     

Effects of immunization with Cryptococcus neoformans cells or cryptococcal culture filtrate antigen on direct anticryptococcal activities of murine T lymphocytes.

Immunizing CBA/J mice with intact Cryptococcus neoformans cells or with a cryptococcal culture filtrate antigen (CneF) induces an anticryptococcal delayed-type hypersensitivity response. Recently, it has been shown that two phenotypically different T-cell populations are responsible for delayed-type hypersensitivity reactivity in mice immunized with intact cryptococcal cells, whereas only one of those populations is present in mice immunized with soluble cryptococcal antigens in complete Freund's adjuvant (CFA). The purpose of this study was to determine if differences occur with regard to direct anticryptococcal activity between T-lymphocyte-enriched populations from mice immunized with intact viable or dead cryptococcal cells and similar cell populations from mice immunized with the soluble cryptococcal culture filtrate antigen, CneF, emulsified in CFA. The percentage of lymphocytes which form conjugates with C. neoformans and the percentage of cryptococcal growth inhibition in vitro are greater with T-lymphocyte-enriched populations from mice sublethally infected with C. neoformans or from mice immunized with intact heat-killed cryptococcal cells in the presence or absence of CFA than with lymphocyte populations from mice immunized with CneF-CFA. Enhanced anticryptococcal activity of T lymphocytes could be induced by immunizing mice with heat-killed C. neoformans cells of serotype A, B, C, or D as well as by immunizing with a similar preparation of an acapsular C. neoformans mutant but not by immunizing with CFA emulsified with CneF prepared from any one of the C. neoformans isolates. These data indicate that the soluble cryptococcal culture filtrate antigens do not induce the same array of functional T lymphocytes as whole cryptococcal cells.     

Genetic control of resistance to Mycobacterium intracellulare infection in mice.

The susceptibilities of various strains of mice to a highly pathogenic strain of Mycobacterium intracellulare, the Mino strain, were determined by intravenous injection of 5 X 10(6) bacteria. CFU were counted on days 1 and 21 of infection. Among 10 strains of mice, C57BL/6, C57BL/10, BALB/c, B10.BR, B10.A, and B10.D2 were susceptible, whereas DBA/2, A/J, CBA, and C3H/He were resistant. In the susceptible mouse strains, the number of bacteria increased during 21 days of infection, whereas no bacterial growth was observed in the resistant strains. Susceptible mice showed weak but positive delayed-type hypersensitivity to M. intracellulare purified protein derivative 20 days after injection of bacteria. Resistant mice developed no delayed-type hypersensitivity. Histological examination showed severe granulomatous lesions in livers or spleens of the susceptible mice after M. intracellulare injection. Analysis of F1 hybrids of susceptible and resistant strains and of F2 and backcross mice showed that the resistance to M. intracellulare seems to be controlled genetically by a single dominant gene. The pattern of distribution of resistance to M. intracellulare among the mouse strains was consistent with that of natural resistance to Mycobacterium bovis to BCG. Thus, resistance to M. intracellulare infection may be regulated by a gene linked to the Bcg gene on chromosome 1.     

Impairment of granulomatous inflammatory response to Histoplasma capsulatum by inhibitors of angiotensin-converting enzyme.

Systemic infection with Histoplasma capsulatum induced a granulomatous inflammatory response in the lymphoreticular organs of C57BL/6 mice that was associated with elevated levels of angiotensin-converting enzyme (ACE) in the spleens. To determine the influence of ACE on the granulomatous response, either captopril or MK 421, two inhibitors of ACE, were administered intraperitoneally to mice 6 h after intravenous injection of H. capsulatum and then daily for 1 week. Each ACE inhibitor sharply reduced ACE activity in the spleens of infected mice. Both drugs worsened the clinical severity of infection and significantly increased the growth of H. capsulatum in livers and spleens of mice infected for 1 week. The histopathological changes in mice given captopril were more severe, with massive infiltrates of macrophages in proximity to large aggregates of yeasts. Conversely, the administration of captopril for 2 weeks during the resolving phases of infection did not slow the healing of the granulomatous lesions, nor did it provoke a relapse of infection. Captopril did not promote the growth of H. capsulatum in artificial medium. This drug was not cytotoxic to peripheral blood leukocytes or to splenic leukocytes from normal and infected mice. Administration of captopril to normal mice for 1 week did not depress the response of splenocytes of concanavalin A or to phytohemagglutinin, nor did it diminish delayed-type hypersensitivity responses in vivo. Finally, captopril did not augment the growth of H. capsulatum within macrophages. Our results suggest that ACE may participate in the regulation of the granulomatous inflammatory response to H. capsulatum and that ACE inhibition impairs the protective effects of granulomatous inflammation during acute H. capsulatum infection.     

Genetic control of Salmonella typhimurium-induced depression of delayed-type hypersensitivity to sheep erythrocytes in mice.

Infection of mice with a temperature-sensitive mutant of Salmonella typhimurium C5TS allowed the survival of genetically susceptible mice. The ability to mount a delayed-type hypersensitivity (DTH) response to sheep erythrocytes during infection with C5TS was studied in various inbred mouse strains, recombinant inbred strains derived from C57BL/6 (susceptible) and A/J (resistant) mice, and C3H congenic mice. Suppression of the DTH response to sheep erythrocytes was found in mice that carried the Itys allele, the H-2b haplotype, or both. These genes are known to increase susceptibility to S. typhimurium infection. In contrast, no DTH response suppression was observed in mouse strains that carried other genes that increased susceptibility to S. typhimurium, e.g., DBA/2 and C3H/HeJ. Apart from a transient suppression in A/J mice, the DTH responses of resistant mice (A/J and CBA) were normal or increased. The DTH response to sheep erythrocytes could be restored in immunodepressed mice by increasing the immunizing dose, suggesting the possible role of activated macrophages in depression of the DTH response.