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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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Impaired antigen-specific CD4 T cell priming in local draining lymph nodes in μMT mice after C.muridarum i.vag. infection.(A) Bacteria burden at lower genital tract of infected WT mice and μMT mice at various time points post infection as measured by vaginal swabs. Error bars show mean bacterial counts ± SEM. WT and μMT mice were infected intravaginally with 1×105C. muridarum. Fourteen days post infection, cells from spleen and DLNs were analyzed. Data shown are representative results of five independent experiments with at least three mice per group. (B) Histograms showing CD44 expression level on total CD4 T cells of infected WT and μMT mice (spleen and DLNs). (C) Total cell number and PmpG-1-specific CD4 T cell number from infected WT and μMT mice (spleen and DLNs). Error bars show mean number ± SEM. *, p<0.05; **, p<0.01. (D) and (E) Splenocytes from infected WT and μMT mice were stained and enriched for PmpG-1-specific CD4 T cells. (D) CD27 and CCR7 expression on CD4+CD44hiPmpG-1303–311:I-Ab+ cells, T-bet expression on CD4+CD44lo, CD4+CD44hi and CD4+CD44hiPmpG-1303–311:I-Ab+ cells were analyzed by flow cytometry. (E) Graphs showing the mean fluorescence intensity (MFI) of T-bet expression as measured by flow cytometry in (D). Error bars show mean MFI ± SEM. *, p<0.05. (F) Splenocytes from infected WT and μMT mice were stimulated ex vivo for 4 h with PMA and ionomycin in the presence of brefeldin A. IFNγ and TNFα production by activated CD4 T cells (CD44hiCD4+) were detected by flow cytometry. The percentages of IFNγ+, TNFα+ and IFNγ+TNFα+ CD4 T cells were summarized in the graph. Error bars show mean number ± SEM.
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ppat-1003707-g004: Impaired antigen-specific CD4 T cell priming in local draining lymph nodes in μMT mice after C.muridarum i.vag. infection.(A) Bacteria burden at lower genital tract of infected WT mice and μMT mice at various time points post infection as measured by vaginal swabs. Error bars show mean bacterial counts ± SEM. WT and μMT mice were infected intravaginally with 1×105C. muridarum. Fourteen days post infection, cells from spleen and DLNs were analyzed. Data shown are representative results of five independent experiments with at least three mice per group. (B) Histograms showing CD44 expression level on total CD4 T cells of infected WT and μMT mice (spleen and DLNs). (C) Total cell number and PmpG-1-specific CD4 T cell number from infected WT and μMT mice (spleen and DLNs). Error bars show mean number ± SEM. *, p<0.05; **, p<0.01. (D) and (E) Splenocytes from infected WT and μMT mice were stained and enriched for PmpG-1-specific CD4 T cells. (D) CD27 and CCR7 expression on CD4+CD44hiPmpG-1303–311:I-Ab+ cells, T-bet expression on CD4+CD44lo, CD4+CD44hi and CD4+CD44hiPmpG-1303–311:I-Ab+ cells were analyzed by flow cytometry. (E) Graphs showing the mean fluorescence intensity (MFI) of T-bet expression as measured by flow cytometry in (D). Error bars show mean MFI ± SEM. *, p<0.05. (F) Splenocytes from infected WT and μMT mice were stimulated ex vivo for 4 h with PMA and ionomycin in the presence of brefeldin A. IFNγ and TNFα production by activated CD4 T cells (CD44hiCD4+) were detected by flow cytometry. The percentages of IFNγ+, TNFα+ and IFNγ+TNFα+ CD4 T cells were summarized in the graph. Error bars show mean number ± SEM.

Mentions: Next, we examined bacterial shedding after vaginal infection of WT and μMT mice with Chlamydia. Consistent with previous reports [17], [18], bacterial shedding was unaffected by the absence of B cells (Fig. 4A). However, more detailed analysis of the local draining lymph nodes of μMT mice suggested significantly reduced activation of CD4 T cells (Fig. 4B). Indeed, using the PmpG-1303–311:I-Ab tetramer, we detected much lower clonal expansion of Chlamydia-specific CD4 T cells in the local draining lymph node of infected μMT mice compared to WT mice (60±12 in μMT mice vs 166±36 in WT mice, p<0.01; Fig. 4C). This reduced local response was also accompanied by dissemination of Chlamydia to the spleen and peritoneal cavity, where ascites was noted 14 days post infection (Fig. 5A and 5B). Analysis of ascites fluid from μMT mice revealed a large proportion of macrophages (F4/80+), monocytes (Gr-1+) and T lymphocytes (Fig. 5C). In addition, PmpG-1-specific CD4 T cells were abundant in ascites, demonstrating that much of the lymphocyte infiltrate into the peritoneal cavity is likely to be Chlamydia-specific (Fig. 5C). Thus, the absence of B cells reduces local CD4 T cell priming and allows bacterial dissemination.


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)

Impaired antigen-specific CD4 T cell priming in local draining lymph nodes in μMT mice after C.muridarum i.vag. infection.(A) Bacteria burden at lower genital tract of infected WT mice and μMT mice at various time points post infection as measured by vaginal swabs. Error bars show mean bacterial counts ± SEM. WT and μMT mice were infected intravaginally with 1×105C. muridarum. Fourteen days post infection, cells from spleen and DLNs were analyzed. Data shown are representative results of five independent experiments with at least three mice per group. (B) Histograms showing CD44 expression level on total CD4 T cells of infected WT and μMT mice (spleen and DLNs). (C) Total cell number and PmpG-1-specific CD4 T cell number from infected WT and μMT mice (spleen and DLNs). Error bars show mean number ± SEM. *, p<0.05; **, p<0.01. (D) and (E) Splenocytes from infected WT and μMT mice were stained and enriched for PmpG-1-specific CD4 T cells. (D) CD27 and CCR7 expression on CD4+CD44hiPmpG-1303–311:I-Ab+ cells, T-bet expression on CD4+CD44lo, CD4+CD44hi and CD4+CD44hiPmpG-1303–311:I-Ab+ cells were analyzed by flow cytometry. (E) Graphs showing the mean fluorescence intensity (MFI) of T-bet expression as measured by flow cytometry in (D). Error bars show mean MFI ± SEM. *, p<0.05. (F) Splenocytes from infected WT and μMT mice were stimulated ex vivo for 4 h with PMA and ionomycin in the presence of brefeldin A. IFNγ and TNFα production by activated CD4 T cells (CD44hiCD4+) were detected by flow cytometry. The percentages of IFNγ+, TNFα+ and IFNγ+TNFα+ CD4 T cells were summarized in the graph. Error bars show mean number ± SEM.
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Related In: Results  -  Collection

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ppat-1003707-g004: Impaired antigen-specific CD4 T cell priming in local draining lymph nodes in μMT mice after C.muridarum i.vag. infection.(A) Bacteria burden at lower genital tract of infected WT mice and μMT mice at various time points post infection as measured by vaginal swabs. Error bars show mean bacterial counts ± SEM. WT and μMT mice were infected intravaginally with 1×105C. muridarum. Fourteen days post infection, cells from spleen and DLNs were analyzed. Data shown are representative results of five independent experiments with at least three mice per group. (B) Histograms showing CD44 expression level on total CD4 T cells of infected WT and μMT mice (spleen and DLNs). (C) Total cell number and PmpG-1-specific CD4 T cell number from infected WT and μMT mice (spleen and DLNs). Error bars show mean number ± SEM. *, p<0.05; **, p<0.01. (D) and (E) Splenocytes from infected WT and μMT mice were stained and enriched for PmpG-1-specific CD4 T cells. (D) CD27 and CCR7 expression on CD4+CD44hiPmpG-1303–311:I-Ab+ cells, T-bet expression on CD4+CD44lo, CD4+CD44hi and CD4+CD44hiPmpG-1303–311:I-Ab+ cells were analyzed by flow cytometry. (E) Graphs showing the mean fluorescence intensity (MFI) of T-bet expression as measured by flow cytometry in (D). Error bars show mean MFI ± SEM. *, p<0.05. (F) Splenocytes from infected WT and μMT mice were stimulated ex vivo for 4 h with PMA and ionomycin in the presence of brefeldin A. IFNγ and TNFα production by activated CD4 T cells (CD44hiCD4+) were detected by flow cytometry. The percentages of IFNγ+, TNFα+ and IFNγ+TNFα+ CD4 T cells were summarized in the graph. Error bars show mean number ± SEM.
Mentions: Next, we examined bacterial shedding after vaginal infection of WT and μMT mice with Chlamydia. Consistent with previous reports [17], [18], bacterial shedding was unaffected by the absence of B cells (Fig. 4A). However, more detailed analysis of the local draining lymph nodes of μMT mice suggested significantly reduced activation of CD4 T cells (Fig. 4B). Indeed, using the PmpG-1303–311:I-Ab tetramer, we detected much lower clonal expansion of Chlamydia-specific CD4 T cells in the local draining lymph node of infected μMT mice compared to WT mice (60±12 in μMT mice vs 166±36 in WT mice, p<0.01; Fig. 4C). This reduced local response was also accompanied by dissemination of Chlamydia to the spleen and peritoneal cavity, where ascites was noted 14 days post infection (Fig. 5A and 5B). Analysis of ascites fluid from μMT mice revealed a large proportion of macrophages (F4/80+), monocytes (Gr-1+) and T lymphocytes (Fig. 5C). In addition, PmpG-1-specific CD4 T cells were abundant in ascites, demonstrating that much of the lymphocyte infiltrate into the peritoneal cavity is likely to be Chlamydia-specific (Fig. 5C). Thus, the absence of B cells reduces local CD4 T cell priming and allows bacterial dissemination.

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