IN THIS ISSUE
http://www.100md.com
免疫学杂志 2005年第8期
Cathelicidin and innate intestinal immunity
The bacterial pathogen Citrobacter rodentium, similar to human enteropathogenic Escherichia coli, causes mucosal erosions, increased epithelial cell proliferation, and crypt hyperplasia in the mouse colon. However, little is known about early innate mucosal defenses against C. rodentium. Iimura et al. (p. 4901 ) found high-level mRNA expression of murine cathelicidin-related antimicrobial peptide (mCRAMP) only in surface colon epithelial cells. The pattern of mCRAMP expression in mice paralleled that of a related cathelicidin in the human colon. Synthetic mCRAMP inhibited growth of C. rodentium and two related human pathogens; colon epithelial cell extracts from wild-type mice were more antimicrobial for C. rodentium than extracts from mice lacking mCRAMP (Cnlp–/–). Colonization and colon pathology at day 7 after an oral inoculum of the bacteria were greater in the Cnlp–/– mice than in wild-type littermates. Although no wild-type mice developed established infections with a 20-fold lower dose of bacteria, Cnlp–/– mice did become infected. Initial surface colonization of colon epithelium and surface epithelial cell damage were greater in the mutant mice after both doses, but the differences disappeared by day 14, at which time wild-type colon epithelium lacked mCRAMP. The data indicate that epithelial cathelicidin is important in the early innate antimicrobial defense against infection of the mouse colon by the foodborne pathogen C. rodentium.
Male-specific thymic involution
Variation in glycosylation patterns among subsets of thymocytes is determined by developmental expression of sialyl transferases; improper surface sialylation in vivo results in thymic apoptosis of CD4+CD8+ T cells. Although androgens have been shown to induce thymic involution, the mechanism by which they act is unknown. Mucci et al. (p. 4545 ) injected mice i.v. with trans-sialidase (TS), a virulence factor shed by Trypanosoma cruzi. Thymocytes in male mice and female thymocytes in a chimeric male mouse were severely depleted 3 days after the last of three injections of TS as measured by thymocyte counts and H&E and TUNEL staining. The TS-induced depletion did not occur in male mice implanted with a pellet releasing a nonsteroidal anti-androgen, in female mice or in gonadectomized male mice. Treatment of female mice or gonadectomized male mice with testosterone 20 days before TS treatment induced thymocyte apoptosis. Male mice deficient for the androgen receptor (AR) did not experience TS-induced apoptosis. Bone marrow from AR– mice transferred into lethally irradiated wild-type males underwent TS-induced apoptosis; no apoptosis was seen in the reciprocal experiment where AR– recipients of wild-type thymocytes were implanted with testosterone pellets. TS treatment did not induce thymocyte apoptosis in male or female mice lacking CD43 surface mucin or TNFR1–/– mice. Increased caspase 3 fluorescence was detected in TS-treated thymus organ cultures. The data support roles for thymocyte CD43, plasma androgens, and the TNFR1 pathway in apoptosis of thymocytes with altered sialylation patterns in male mice.
Generating high-affinity mAbs in vivo
Expression of germinal center-associated nuclear protein (GANP) is elevated in germinal center B cells after in vivo immunization of mice with a T cell-dependent Ag (TD-Ag). Moreover, B-ganp–/– mice have defective high-affinity Ab responses to TD-Ag. To define the molecular mechanism for GANP function in high-affinity Ab responses, Sakaguchi et al. (p. 4485 ) created Ganp transgenic mice (GanpTg) with a 2-fold greater expression of ganp transcripts in B cells and heightened B cell responses to CD40 stimulation. Increases in Ab affinity during a secondary response against a TD-Ag were found by RT-PCR to occur through mutation in a particular VH locus in splenic B cells. Hybridomas secreting high-affinity mAbs were cloned, and binding constants were measured by the BIAcore system with a sensor chip conjugated with the immunizing Ag. DNA sequence analysis of genomic DNAs indicated greatly increased mutation of the same VH region but few VL region mutations among the hybridomas. Established B cell hybridomas from GanpTg mice immunized with specific peptides of the HIV-1 major glycoprotein produced mAbs of much greater affinity compared with mAbs produced by conventional methods. Mutation in one specific VH region predominated among the high-affinity mAbs. The anti-HIV-1 mAbs had higher binding to virus epitope-expressing cells and greater virus-neutralizing activity compared with conventional anti-HIV-1 mAbs. The results confirm that GANP is involved in generation of high-affinity Ab in vivo and that immunizing GanpTg mice can be used to generate high-affinity mAbs for a variety of purposes.
Apoptosis in patients with sepsis
Immunosuppression in patients with sepsis is due to depletion of immune effector cells in combination with increased numbers of apoptotic cells. However, little is known about apoptosis in circulating lymphocytes. Hotchkiss et al. (p. 5110 ) found elevated levels of IL-10, IFN-, and TNF-, and a decreased percentage of CD4+ T cells in patients with sepsis compared with critically ill nonseptic patients. Both groups of ill patients (71 septic and 55 nonseptic) had a lower percentage of CD3+ T cells compared with healthy controls. Lymphocyte apoptosis, measured by forward and side scatter in flow cytometry and staining for apoptosis markers, was higher only in patients with sepsis, with or without septic shock. CD3+ T cell apoptosis levels were highest during episodes of sepsis, and CD3+ T cells were positive for active caspases 3, 8, and 9 by flow cytometry or by immunoblot analysis. CD3+ T cell apoptosis fell with resolution of sepsis, and active caspases were rarely detected in critically ill nonseptic patients or in healthy volunteers. Increased active caspase 3 was detected by flow cytometry in lymphocyte populations with normal or decreased Bcl-2 levels. The authors conclude that both death receptor- and mitochondrial-mediated apoptotic pathways occur in different CD3+ T cell populations in patients with sepsis.
Improving vaccine design
One approach to developing more effective antitumor vaccines has been to create an "anchor-fixed" altered peptide ligand (APL) of tumor-associated Ags. However, the molecular basis for the enhanced immunogenicity of APLs is unknown. Borbulevych et al. (p. 4812 ) compared the crystal structures of HLA-A2 bound with the 209–217 peptide of the melanoma gp100 membrane glycoprotein (gp100209/HLA-A2) with its "anchor-fixed" variant in which methionine replaced threonine at position 2 (T2M-modified gp100209/HLA-A2). Although the T2M-modified peptide has been shown clinically to increase immunogenicity, the x-ray data did not show structural differences in peptide formation, in the HLA-A2 helices, or in the overall size and shape of the P2 pocket in which the peptides bound. ELISPOT measurements of IFN- release by Ag-specific T cells from 106 PBMC samples of 95 patients at high risk for recurrence of melanoma who had been vaccinated with the T2M-modified peptide did not reveal any differences with native vs modified peptide. Steady-state fluorescence anisotropy, used to measure peptide dissociation of soluble, purified peptide/HLA-A2 complexes, showed that the T2M-modified peptide had a slower dissociation rate than the native peptide. The authors suggest that the enhanced stability of the T2M-modified peptide/HLA-A2 complex is a consequence of the amino acid substitution in the APL and validates the anchor-fixing approach to vaccine design.
Fas/FasL and fetal tolerance
Peripheral T cell tolerance is required to prevent excessive T cell reactivity to Ags that are expressed developmentally. Vacchio and Hodes have shown that CD28/B7 interactions are required for tolerance induction in CD8+ T cells during pregnancy in mice. On p. 4657 , the same authors examined the role of Fas and FasL interactions in development of maternal T cell tolerance of the fetus. H-Y TCR transgenic wild-type females carrying male fetuses had a 50% reduction of spleen and lymph node H-Y-specific T cells on day 18 of pregnancy compared with pregnant H-Y TCR transgenic females expressing nonfunctional Fas or nonfunctional FasL or nonpregnant controls. No deletion of H-Y-specific T cells occurred in pregnancies with only female fetuses. CFSE-labeled spleen cells from day 18 pregnant H-Y mice were hyporesponsive to proliferation in vitro when exposed to male APCs. Transfer of embryos from wild-type H-Y pregnancies, but not of embryos from nonfunctional FasL H-Y pregnancies, into pseudopregnant wild-type
H-Y females resulted in decreased numbers of H-Y-specific T cells in the spleen of the recipient. In the reverse experiment, deletion of peripheral T cells and hyporesponsiveness of remaining cells occurred only when H-Y females deficient in functional FasL were bred to wild-type males; no peripheral T cell deletion or hyporesponsiveness was seen when both breeding partners lacked functional FasL. The experiments show that interaction of Fas-expressing maternal T cells with FasL-expressing fetal cells induces deletion and hyporesponsiveness of H-Y-specific T cells during pregnancy.
IL-2 promoter modifications in activated CD4+ T cells
Expression of the IL-2 gene in activated CD4+ T cells is dependent on CD28 costimulation. Simultaneous activation of enzymes that modify nucleosomes and remodel chromatin suggests that those enzymes might act on the IL-2 promoter. Thomas et al. (p. 4639 ) PCR-amplified DNA of chromatin immunoprecipitation complexes obtained using an Ab specific for acetylated histone H3. Strong histone acetylation upstream of the IL-2 gene was seen in murine CD4+ T cells activated in the presence of CD28 costimulation, whereas little or no histone acetylation was detected in naive CD4+ T cells or in CD4+ T cells rendered anergic by activation in the absence of CD28 costimulation. Most of the acetylation occurred within 24 h in the minimal promoter/enhancer region, peaked at 48 h, and persisted to 72 h. The IL-2 promoter in nuclei isolated from effector CD4+ T cells, but not in nuclei from naive or anergic CD4+ T cells, was accessible to nuclease digestion. The most promoter-proximal genomic regions remained accessible during a 3-day response, but more distal regions (up to 700 bp upstream) lost accessibility. Approximately 35% of CpG dinucleotides in the naive IL-2 promoter/enhancer were found to be methylated; the most proximal CpG site at –68 bp was methylated to 80%. The percentages decreased greatly in effector CD4+ T cells costimulated with CD28 for 3 days, but no significant change was seen in cells stimulated in the absence of CD28 costimulation. The experiments demonstrate that epigenetic modifications to the IL-2 promoter in CD28-costimulated CD4+ T cells involve acetylation, chromatin remodeling, and demethylation.
Summaries written by Dorothy L. Buchhagen, Ph.D.
Related articles in The JI:
Generation of High-Affinity Antibody against T Cell-Dependent Antigen in the Ganp Gene-Transgenic Mouse
Nobuo Sakaguchi, Tetsuya Kimura, Shuzo Matsushita, Satoru Fujimura, Junji Shibata, Masatake Araki, Tamami Sakamoto, Chiemi Minoda, and Kazuhiko Kuwahara
The JI 2005 174: 4485-4494.
A Sexual Dimorphism in Intrathymic Sialylation Survey Is Revealed by the trans-Sialidase from Trypanosoma cruzi
Juan Mucci, Esteban Mocetti, María Susana Leguizamón, and Oscar Campetella
The JI 2005 174: 4545-4550.
Signals from CD28 Induce Stable Epigenetic Modification of the IL-2 Promoter
Rajan M. Thomas, Ling Gao, and Andrew D. Wells
The JI 2005 174: 4639-4646.
Fetal Expression of Fas Ligand Is Necessary and Sufficient for Induction of CD8 T Cell Tolerance to the Fetal Antigen H-Y during Pregnancy
Melanie S. Vacchio and Richard J. Hodes
The JI 2005 174: 4657-4661.
Increased Immunogenicity of an Anchor-Modified Tumor-Associated Antigen Is Due to the Enhanced Stability of the Peptide/MHC Complex: Implications for Vaccine Design
Oleg Y. Borbulevych, Tiffany K. Baxter, Zhiya Yu, Nicholas P. Restifo, and Brian M. Baker
The JI 2005 174: 4812-4820.
Cathelicidin Mediates Innate Intestinal Defense against Colonization with Epithelial Adherent Bacterial Pathogens
Mitsutoshi Iimura, Richard L. Gallo, Koji Hase, Yukiko Miyamoto, Lars Eckmann, and Martin F. Kagnoff
The JI 2005 174: 4901-4907.
Accelerated Lymphocyte Death in Sepsis Occurs by both the Death Receptor and Mitochondrial Pathways
Richard S. Hotchkiss, Stephen B. Osmon, Katherine C. Chang, Tracey H. Wagner, Craig M. Coopersmith, and Irene E. Karl
The JI 2005 174: 5110-5118.
The bacterial pathogen Citrobacter rodentium, similar to human enteropathogenic Escherichia coli, causes mucosal erosions, increased epithelial cell proliferation, and crypt hyperplasia in the mouse colon. However, little is known about early innate mucosal defenses against C. rodentium. Iimura et al. (p. 4901 ) found high-level mRNA expression of murine cathelicidin-related antimicrobial peptide (mCRAMP) only in surface colon epithelial cells. The pattern of mCRAMP expression in mice paralleled that of a related cathelicidin in the human colon. Synthetic mCRAMP inhibited growth of C. rodentium and two related human pathogens; colon epithelial cell extracts from wild-type mice were more antimicrobial for C. rodentium than extracts from mice lacking mCRAMP (Cnlp–/–). Colonization and colon pathology at day 7 after an oral inoculum of the bacteria were greater in the Cnlp–/– mice than in wild-type littermates. Although no wild-type mice developed established infections with a 20-fold lower dose of bacteria, Cnlp–/– mice did become infected. Initial surface colonization of colon epithelium and surface epithelial cell damage were greater in the mutant mice after both doses, but the differences disappeared by day 14, at which time wild-type colon epithelium lacked mCRAMP. The data indicate that epithelial cathelicidin is important in the early innate antimicrobial defense against infection of the mouse colon by the foodborne pathogen C. rodentium.
Male-specific thymic involution
Variation in glycosylation patterns among subsets of thymocytes is determined by developmental expression of sialyl transferases; improper surface sialylation in vivo results in thymic apoptosis of CD4+CD8+ T cells. Although androgens have been shown to induce thymic involution, the mechanism by which they act is unknown. Mucci et al. (p. 4545 ) injected mice i.v. with trans-sialidase (TS), a virulence factor shed by Trypanosoma cruzi. Thymocytes in male mice and female thymocytes in a chimeric male mouse were severely depleted 3 days after the last of three injections of TS as measured by thymocyte counts and H&E and TUNEL staining. The TS-induced depletion did not occur in male mice implanted with a pellet releasing a nonsteroidal anti-androgen, in female mice or in gonadectomized male mice. Treatment of female mice or gonadectomized male mice with testosterone 20 days before TS treatment induced thymocyte apoptosis. Male mice deficient for the androgen receptor (AR) did not experience TS-induced apoptosis. Bone marrow from AR– mice transferred into lethally irradiated wild-type males underwent TS-induced apoptosis; no apoptosis was seen in the reciprocal experiment where AR– recipients of wild-type thymocytes were implanted with testosterone pellets. TS treatment did not induce thymocyte apoptosis in male or female mice lacking CD43 surface mucin or TNFR1–/– mice. Increased caspase 3 fluorescence was detected in TS-treated thymus organ cultures. The data support roles for thymocyte CD43, plasma androgens, and the TNFR1 pathway in apoptosis of thymocytes with altered sialylation patterns in male mice.
Generating high-affinity mAbs in vivo
Expression of germinal center-associated nuclear protein (GANP) is elevated in germinal center B cells after in vivo immunization of mice with a T cell-dependent Ag (TD-Ag). Moreover, B-ganp–/– mice have defective high-affinity Ab responses to TD-Ag. To define the molecular mechanism for GANP function in high-affinity Ab responses, Sakaguchi et al. (p. 4485 ) created Ganp transgenic mice (GanpTg) with a 2-fold greater expression of ganp transcripts in B cells and heightened B cell responses to CD40 stimulation. Increases in Ab affinity during a secondary response against a TD-Ag were found by RT-PCR to occur through mutation in a particular VH locus in splenic B cells. Hybridomas secreting high-affinity mAbs were cloned, and binding constants were measured by the BIAcore system with a sensor chip conjugated with the immunizing Ag. DNA sequence analysis of genomic DNAs indicated greatly increased mutation of the same VH region but few VL region mutations among the hybridomas. Established B cell hybridomas from GanpTg mice immunized with specific peptides of the HIV-1 major glycoprotein produced mAbs of much greater affinity compared with mAbs produced by conventional methods. Mutation in one specific VH region predominated among the high-affinity mAbs. The anti-HIV-1 mAbs had higher binding to virus epitope-expressing cells and greater virus-neutralizing activity compared with conventional anti-HIV-1 mAbs. The results confirm that GANP is involved in generation of high-affinity Ab in vivo and that immunizing GanpTg mice can be used to generate high-affinity mAbs for a variety of purposes.
Apoptosis in patients with sepsis
Immunosuppression in patients with sepsis is due to depletion of immune effector cells in combination with increased numbers of apoptotic cells. However, little is known about apoptosis in circulating lymphocytes. Hotchkiss et al. (p. 5110 ) found elevated levels of IL-10, IFN-, and TNF-, and a decreased percentage of CD4+ T cells in patients with sepsis compared with critically ill nonseptic patients. Both groups of ill patients (71 septic and 55 nonseptic) had a lower percentage of CD3+ T cells compared with healthy controls. Lymphocyte apoptosis, measured by forward and side scatter in flow cytometry and staining for apoptosis markers, was higher only in patients with sepsis, with or without septic shock. CD3+ T cell apoptosis levels were highest during episodes of sepsis, and CD3+ T cells were positive for active caspases 3, 8, and 9 by flow cytometry or by immunoblot analysis. CD3+ T cell apoptosis fell with resolution of sepsis, and active caspases were rarely detected in critically ill nonseptic patients or in healthy volunteers. Increased active caspase 3 was detected by flow cytometry in lymphocyte populations with normal or decreased Bcl-2 levels. The authors conclude that both death receptor- and mitochondrial-mediated apoptotic pathways occur in different CD3+ T cell populations in patients with sepsis.
Improving vaccine design
One approach to developing more effective antitumor vaccines has been to create an "anchor-fixed" altered peptide ligand (APL) of tumor-associated Ags. However, the molecular basis for the enhanced immunogenicity of APLs is unknown. Borbulevych et al. (p. 4812 ) compared the crystal structures of HLA-A2 bound with the 209–217 peptide of the melanoma gp100 membrane glycoprotein (gp100209/HLA-A2) with its "anchor-fixed" variant in which methionine replaced threonine at position 2 (T2M-modified gp100209/HLA-A2). Although the T2M-modified peptide has been shown clinically to increase immunogenicity, the x-ray data did not show structural differences in peptide formation, in the HLA-A2 helices, or in the overall size and shape of the P2 pocket in which the peptides bound. ELISPOT measurements of IFN- release by Ag-specific T cells from 106 PBMC samples of 95 patients at high risk for recurrence of melanoma who had been vaccinated with the T2M-modified peptide did not reveal any differences with native vs modified peptide. Steady-state fluorescence anisotropy, used to measure peptide dissociation of soluble, purified peptide/HLA-A2 complexes, showed that the T2M-modified peptide had a slower dissociation rate than the native peptide. The authors suggest that the enhanced stability of the T2M-modified peptide/HLA-A2 complex is a consequence of the amino acid substitution in the APL and validates the anchor-fixing approach to vaccine design.
Fas/FasL and fetal tolerance
Peripheral T cell tolerance is required to prevent excessive T cell reactivity to Ags that are expressed developmentally. Vacchio and Hodes have shown that CD28/B7 interactions are required for tolerance induction in CD8+ T cells during pregnancy in mice. On p. 4657 , the same authors examined the role of Fas and FasL interactions in development of maternal T cell tolerance of the fetus. H-Y TCR transgenic wild-type females carrying male fetuses had a 50% reduction of spleen and lymph node H-Y-specific T cells on day 18 of pregnancy compared with pregnant H-Y TCR transgenic females expressing nonfunctional Fas or nonfunctional FasL or nonpregnant controls. No deletion of H-Y-specific T cells occurred in pregnancies with only female fetuses. CFSE-labeled spleen cells from day 18 pregnant H-Y mice were hyporesponsive to proliferation in vitro when exposed to male APCs. Transfer of embryos from wild-type H-Y pregnancies, but not of embryos from nonfunctional FasL H-Y pregnancies, into pseudopregnant wild-type
H-Y females resulted in decreased numbers of H-Y-specific T cells in the spleen of the recipient. In the reverse experiment, deletion of peripheral T cells and hyporesponsiveness of remaining cells occurred only when H-Y females deficient in functional FasL were bred to wild-type males; no peripheral T cell deletion or hyporesponsiveness was seen when both breeding partners lacked functional FasL. The experiments show that interaction of Fas-expressing maternal T cells with FasL-expressing fetal cells induces deletion and hyporesponsiveness of H-Y-specific T cells during pregnancy.
IL-2 promoter modifications in activated CD4+ T cells
Expression of the IL-2 gene in activated CD4+ T cells is dependent on CD28 costimulation. Simultaneous activation of enzymes that modify nucleosomes and remodel chromatin suggests that those enzymes might act on the IL-2 promoter. Thomas et al. (p. 4639 ) PCR-amplified DNA of chromatin immunoprecipitation complexes obtained using an Ab specific for acetylated histone H3. Strong histone acetylation upstream of the IL-2 gene was seen in murine CD4+ T cells activated in the presence of CD28 costimulation, whereas little or no histone acetylation was detected in naive CD4+ T cells or in CD4+ T cells rendered anergic by activation in the absence of CD28 costimulation. Most of the acetylation occurred within 24 h in the minimal promoter/enhancer region, peaked at 48 h, and persisted to 72 h. The IL-2 promoter in nuclei isolated from effector CD4+ T cells, but not in nuclei from naive or anergic CD4+ T cells, was accessible to nuclease digestion. The most promoter-proximal genomic regions remained accessible during a 3-day response, but more distal regions (up to 700 bp upstream) lost accessibility. Approximately 35% of CpG dinucleotides in the naive IL-2 promoter/enhancer were found to be methylated; the most proximal CpG site at –68 bp was methylated to 80%. The percentages decreased greatly in effector CD4+ T cells costimulated with CD28 for 3 days, but no significant change was seen in cells stimulated in the absence of CD28 costimulation. The experiments demonstrate that epigenetic modifications to the IL-2 promoter in CD28-costimulated CD4+ T cells involve acetylation, chromatin remodeling, and demethylation.
Summaries written by Dorothy L. Buchhagen, Ph.D.
Related articles in The JI:
Generation of High-Affinity Antibody against T Cell-Dependent Antigen in the Ganp Gene-Transgenic Mouse
Nobuo Sakaguchi, Tetsuya Kimura, Shuzo Matsushita, Satoru Fujimura, Junji Shibata, Masatake Araki, Tamami Sakamoto, Chiemi Minoda, and Kazuhiko Kuwahara
The JI 2005 174: 4485-4494.
A Sexual Dimorphism in Intrathymic Sialylation Survey Is Revealed by the trans-Sialidase from Trypanosoma cruzi
Juan Mucci, Esteban Mocetti, María Susana Leguizamón, and Oscar Campetella
The JI 2005 174: 4545-4550.
Signals from CD28 Induce Stable Epigenetic Modification of the IL-2 Promoter
Rajan M. Thomas, Ling Gao, and Andrew D. Wells
The JI 2005 174: 4639-4646.
Fetal Expression of Fas Ligand Is Necessary and Sufficient for Induction of CD8 T Cell Tolerance to the Fetal Antigen H-Y during Pregnancy
Melanie S. Vacchio and Richard J. Hodes
The JI 2005 174: 4657-4661.
Increased Immunogenicity of an Anchor-Modified Tumor-Associated Antigen Is Due to the Enhanced Stability of the Peptide/MHC Complex: Implications for Vaccine Design
Oleg Y. Borbulevych, Tiffany K. Baxter, Zhiya Yu, Nicholas P. Restifo, and Brian M. Baker
The JI 2005 174: 4812-4820.
Cathelicidin Mediates Innate Intestinal Defense against Colonization with Epithelial Adherent Bacterial Pathogens
Mitsutoshi Iimura, Richard L. Gallo, Koji Hase, Yukiko Miyamoto, Lars Eckmann, and Martin F. Kagnoff
The JI 2005 174: 4901-4907.
Accelerated Lymphocyte Death in Sepsis Occurs by both the Death Receptor and Mitochondrial Pathways
Richard S. Hotchkiss, Stephen B. Osmon, Katherine C. Chang, Tracey H. Wagner, Craig M. Coopersmith, and Irene E. Karl
The JI 2005 174: 5110-5118.