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B cells enhance antigen-specific CD4 T cell priming and prevent bacteria dissemination following Chlamydia muridarum genital tract infection.

Li LX, McSorley SJ - PLoS Pathog. (2013)

Bottom Line: While most lymph node Chlamydia-specific CD4 T cells expressed T-bet, a small percentage co-expressed Foxp3, and RORγt-expressing T cells were enriched within the reproductive tract.Local Chlamydia-specific CD4 T cell priming was markedly reduced in mice lacking B cells, and bacteria were able to disseminate to the peritoneal cavity, initiating a cellular infiltrate and ascites.However, bacterial dissemination also coincided with elevated systemic Chlamydia-specific CD4 T cell responses and resolution of primary infection.

View Article: PubMed Central - PubMed

Affiliation: Center for Comparative Medicine, Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, Davis, California, United States of America.

ABSTRACT
B cells can contribute to acquired immunity against intracellular bacteria, but do not usually participate in primary clearance. Here, we examined the endogenous CD4 T cell response to genital infection with Chlamydia muridarum using MHC class-II tetramers. Chlamydia-specific CD4 T cells expanded rapidly and persisted as a stable memory pool for several months after infection. While most lymph node Chlamydia-specific CD4 T cells expressed T-bet, a small percentage co-expressed Foxp3, and RORγt-expressing T cells were enriched within the reproductive tract. Local Chlamydia-specific CD4 T cell priming was markedly reduced in mice lacking B cells, and bacteria were able to disseminate to the peritoneal cavity, initiating a cellular infiltrate and ascites. However, bacterial dissemination also coincided with elevated systemic Chlamydia-specific CD4 T cell responses and resolution of primary infection. Together, these data reveal heterogeneity in pathogen-specific CD4 T cell responses within the genital tract and an unexpected requirement for B cells in regulating local T cell activation and bacterial dissemination during genital infection.

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Related in: MedlinePlus

Kinetics of antigen-specific CD4+ T cell expansion after C. muridarum intravaginal (i.vag.) infection.(A) C57BL/6 mice were immunized subcutaneously with 100 µg of C. muridarum peptide (RplF51–59, Aasf24–32 or PmpG-1303–311) in the presence of CFA or infected intravenously with either 1×105C. muridarum or 5×105S. typhimurium. Seven days post immunization/infection, axillary, brachial and inguinal lymph nodes were isolated from peptide-immunized mice, spleen and lymph nodes were harvested from C. muridarum or S. Typhimurium infected mice and endogenous RplF, Aasf or PmpG-1-specific CD4 T cells were detected with RplF51–59:I-Ab, Aasf24–32:I-Ab or PmpG-1303–311:I-Ab tetramers, respectively. FACS plots showing representative results of tetramer staining after fluorescent microbeads enrichment. All plots were pre-gated on CD11b−CD11c−F4/80−B220−CD3+CD4+ cells except CD8+ T cells (bottom row), which were gated on CD11b−CD11c−F4/80−B220−CD3+CD8+ cells. (B–D) C57BL/6 mice were infected i.vag. with 1×105C. muridarum. (B) At various time points post infection, bacteria burden at lower genital tract was measured by vaginal swabs. (C) Endogenous PmpG-1-specific CD4 T cells in the spleen, draining lymph nodes and non-draining lymph nodes were detected by PmpG-1303–311:I-Ab tetramers. FACS plots showing representative results of tetramer staining after magnetic enrichment at day 0, 7, 14 and 21 days post infection. All plots were pre-gated on CD11b−CD11c−F4/80−B220−CD3+CD4+ cells. (D) Total cell number of endogenous PmpG-1-specific CD4 T cells calculated based on FACS analysis. Data shown are representative results from two independent experiments with three to five mice per time point. Bar graphs show mean number ± SEM.
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ppat-1003707-g002: Kinetics of antigen-specific CD4+ T cell expansion after C. muridarum intravaginal (i.vag.) infection.(A) C57BL/6 mice were immunized subcutaneously with 100 µg of C. muridarum peptide (RplF51–59, Aasf24–32 or PmpG-1303–311) in the presence of CFA or infected intravenously with either 1×105C. muridarum or 5×105S. typhimurium. Seven days post immunization/infection, axillary, brachial and inguinal lymph nodes were isolated from peptide-immunized mice, spleen and lymph nodes were harvested from C. muridarum or S. Typhimurium infected mice and endogenous RplF, Aasf or PmpG-1-specific CD4 T cells were detected with RplF51–59:I-Ab, Aasf24–32:I-Ab or PmpG-1303–311:I-Ab tetramers, respectively. FACS plots showing representative results of tetramer staining after fluorescent microbeads enrichment. All plots were pre-gated on CD11b−CD11c−F4/80−B220−CD3+CD4+ cells except CD8+ T cells (bottom row), which were gated on CD11b−CD11c−F4/80−B220−CD3+CD8+ cells. (B–D) C57BL/6 mice were infected i.vag. with 1×105C. muridarum. (B) At various time points post infection, bacteria burden at lower genital tract was measured by vaginal swabs. (C) Endogenous PmpG-1-specific CD4 T cells in the spleen, draining lymph nodes and non-draining lymph nodes were detected by PmpG-1303–311:I-Ab tetramers. FACS plots showing representative results of tetramer staining after magnetic enrichment at day 0, 7, 14 and 21 days post infection. All plots were pre-gated on CD11b−CD11c−F4/80−B220−CD3+CD4+ cells. (D) Total cell number of endogenous PmpG-1-specific CD4 T cells calculated based on FACS analysis. Data shown are representative results from two independent experiments with three to five mice per time point. Bar graphs show mean number ± SEM.

Mentions: Previous studies have demonstrated that pMHC class-II tetramers can be used in conjunction with tetramer enrichment, to visualize low frequency endogenous antigen-specific CD4 T cells in infected and immunized mice [22], [23]. We constructed three distinct pMHC class-II tetramers, containing I-Ab with a Chlamydia-specific epitope (RplF51–59, Aasf24–32, or PmpG-1303–311) bound to the MHC class-II β chain. Uninfected C57BL/6 mice contained low frequency antigen-specific CD4 T cell population specific for each Chlamydia epitope (Fig. 2A). However, in mice immunized subcutaneously with peptide/CFA, or infected intravenously with C. muridarum, an expanded population of CD44hi CD4 T cells was detectable 7 days post immunization or infection (Fig. 2A). Tetramer staining was specific, as infection with Salmonella Typhimurium did not induce expansion of tetramer-specific CD4 T cells (Fig. 2A). Furthermore, no CD8 T cells were detected that bound to Chlamydia tetramers (Fig. 2A) Together, these results demonstrate that all three tetramers, RplF51–59:I-Ab, Aasf24–32:I-Ab, and PmpG-1303–311:I-Ab can be used to detect endogenous C. muridarum-CD4 T cells in vivo.


B cells enhance antigen-specific CD4 T cell priming and prevent bacteria dissemination following Chlamydia muridarum genital tract infection.

Li LX, McSorley SJ - PLoS Pathog. (2013)

Kinetics of antigen-specific CD4+ T cell expansion after C. muridarum intravaginal (i.vag.) infection.(A) C57BL/6 mice were immunized subcutaneously with 100 µg of C. muridarum peptide (RplF51–59, Aasf24–32 or PmpG-1303–311) in the presence of CFA or infected intravenously with either 1×105C. muridarum or 5×105S. typhimurium. Seven days post immunization/infection, axillary, brachial and inguinal lymph nodes were isolated from peptide-immunized mice, spleen and lymph nodes were harvested from C. muridarum or S. Typhimurium infected mice and endogenous RplF, Aasf or PmpG-1-specific CD4 T cells were detected with RplF51–59:I-Ab, Aasf24–32:I-Ab or PmpG-1303–311:I-Ab tetramers, respectively. FACS plots showing representative results of tetramer staining after fluorescent microbeads enrichment. All plots were pre-gated on CD11b−CD11c−F4/80−B220−CD3+CD4+ cells except CD8+ T cells (bottom row), which were gated on CD11b−CD11c−F4/80−B220−CD3+CD8+ cells. (B–D) C57BL/6 mice were infected i.vag. with 1×105C. muridarum. (B) At various time points post infection, bacteria burden at lower genital tract was measured by vaginal swabs. (C) Endogenous PmpG-1-specific CD4 T cells in the spleen, draining lymph nodes and non-draining lymph nodes were detected by PmpG-1303–311:I-Ab tetramers. FACS plots showing representative results of tetramer staining after magnetic enrichment at day 0, 7, 14 and 21 days post infection. All plots were pre-gated on CD11b−CD11c−F4/80−B220−CD3+CD4+ cells. (D) Total cell number of endogenous PmpG-1-specific CD4 T cells calculated based on FACS analysis. Data shown are representative results from two independent experiments with three to five mice per time point. Bar graphs show mean number ± SEM.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814678&req=5

ppat-1003707-g002: Kinetics of antigen-specific CD4+ T cell expansion after C. muridarum intravaginal (i.vag.) infection.(A) C57BL/6 mice were immunized subcutaneously with 100 µg of C. muridarum peptide (RplF51–59, Aasf24–32 or PmpG-1303–311) in the presence of CFA or infected intravenously with either 1×105C. muridarum or 5×105S. typhimurium. Seven days post immunization/infection, axillary, brachial and inguinal lymph nodes were isolated from peptide-immunized mice, spleen and lymph nodes were harvested from C. muridarum or S. Typhimurium infected mice and endogenous RplF, Aasf or PmpG-1-specific CD4 T cells were detected with RplF51–59:I-Ab, Aasf24–32:I-Ab or PmpG-1303–311:I-Ab tetramers, respectively. FACS plots showing representative results of tetramer staining after fluorescent microbeads enrichment. All plots were pre-gated on CD11b−CD11c−F4/80−B220−CD3+CD4+ cells except CD8+ T cells (bottom row), which were gated on CD11b−CD11c−F4/80−B220−CD3+CD8+ cells. (B–D) C57BL/6 mice were infected i.vag. with 1×105C. muridarum. (B) At various time points post infection, bacteria burden at lower genital tract was measured by vaginal swabs. (C) Endogenous PmpG-1-specific CD4 T cells in the spleen, draining lymph nodes and non-draining lymph nodes were detected by PmpG-1303–311:I-Ab tetramers. FACS plots showing representative results of tetramer staining after magnetic enrichment at day 0, 7, 14 and 21 days post infection. All plots were pre-gated on CD11b−CD11c−F4/80−B220−CD3+CD4+ cells. (D) Total cell number of endogenous PmpG-1-specific CD4 T cells calculated based on FACS analysis. Data shown are representative results from two independent experiments with three to five mice per time point. Bar graphs show mean number ± SEM.
Mentions: Previous studies have demonstrated that pMHC class-II tetramers can be used in conjunction with tetramer enrichment, to visualize low frequency endogenous antigen-specific CD4 T cells in infected and immunized mice [22], [23]. We constructed three distinct pMHC class-II tetramers, containing I-Ab with a Chlamydia-specific epitope (RplF51–59, Aasf24–32, or PmpG-1303–311) bound to the MHC class-II β chain. Uninfected C57BL/6 mice contained low frequency antigen-specific CD4 T cell population specific for each Chlamydia epitope (Fig. 2A). However, in mice immunized subcutaneously with peptide/CFA, or infected intravenously with C. muridarum, an expanded population of CD44hi CD4 T cells was detectable 7 days post immunization or infection (Fig. 2A). Tetramer staining was specific, as infection with Salmonella Typhimurium did not induce expansion of tetramer-specific CD4 T cells (Fig. 2A). Furthermore, no CD8 T cells were detected that bound to Chlamydia tetramers (Fig. 2A) Together, these results demonstrate that all three tetramers, RplF51–59:I-Ab, Aasf24–32:I-Ab, and PmpG-1303–311:I-Ab can be used to detect endogenous C. muridarum-CD4 T cells in vivo.

Bottom Line: While most lymph node Chlamydia-specific CD4 T cells expressed T-bet, a small percentage co-expressed Foxp3, and RORγt-expressing T cells were enriched within the reproductive tract.Local Chlamydia-specific CD4 T cell priming was markedly reduced in mice lacking B cells, and bacteria were able to disseminate to the peritoneal cavity, initiating a cellular infiltrate and ascites.However, bacterial dissemination also coincided with elevated systemic Chlamydia-specific CD4 T cell responses and resolution of primary infection.

View Article: PubMed Central - PubMed

Affiliation: Center for Comparative Medicine, Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, Davis, California, United States of America.

ABSTRACT
B cells can contribute to acquired immunity against intracellular bacteria, but do not usually participate in primary clearance. Here, we examined the endogenous CD4 T cell response to genital infection with Chlamydia muridarum using MHC class-II tetramers. Chlamydia-specific CD4 T cells expanded rapidly and persisted as a stable memory pool for several months after infection. While most lymph node Chlamydia-specific CD4 T cells expressed T-bet, a small percentage co-expressed Foxp3, and RORγt-expressing T cells were enriched within the reproductive tract. Local Chlamydia-specific CD4 T cell priming was markedly reduced in mice lacking B cells, and bacteria were able to disseminate to the peritoneal cavity, initiating a cellular infiltrate and ascites. However, bacterial dissemination also coincided with elevated systemic Chlamydia-specific CD4 T cell responses and resolution of primary infection. Together, these data reveal heterogeneity in pathogen-specific CD4 T cell responses within the genital tract and an unexpected requirement for B cells in regulating local T cell activation and bacterial dissemination during genital infection.

Show MeSH
Related in: MedlinePlus