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B cell: T cell interactions occur within hepatic granulomas during experimental visceral leishmaniasis.

Moore JW, Beattie L, Dalton JE, Owens BM, Maroof A, Coles MC, Kaye PM - PLoS ONE (2012)

Bottom Line: In infected mice, there was a small increase in mIgM(lo)mIgD(+) mature B2 cells, but no enrichment of B cells with regulatory phenotype or function compared to the naïve hepatic B cell population, as assessed by CD1d and CD5 expression and by IL-10 production.Intra-vital dynamic imaging was used to demonstrate that within the polyclonal B cell population obtained from L. donovani-infected mice, the frequency of B cells that made multiple long contacts with endogenous T cells was greater than that observed using HEL-specific B cells obtained from the same inflammatory environment.These data indicate, therefore, that a subset of this polyclonal B cell population is capable of making cognate interactions with T cells within this unique environment, and provide the first insights into the dynamics of B cells within an inflammatory site.

View Article: PubMed Central - PubMed

Affiliation: Centre for Immunology and Infection, Hull York Medical School and Department of Biology, University of York, Heslington, York, United Kingdom.

ABSTRACT
Hepatic resistance to Leishmania donovani infection in mice is associated with the development of granulomas, in which a variety of lymphoid and non-lymphoid populations accumulate. Although previous studies have identified B cells in hepatic granulomas and functional studies in B cell-deficient mice have suggested a role for B cells in the control of experimental visceral leishmaniasis, little is known about the behaviour of B cells in the granuloma microenvironment. Here, we first compared the hepatic B cell population in infected mice, where ≈60% of B cells are located within granulomas, with that of naïve mice. In infected mice, there was a small increase in mIgM(lo)mIgD(+) mature B2 cells, but no enrichment of B cells with regulatory phenotype or function compared to the naïve hepatic B cell population, as assessed by CD1d and CD5 expression and by IL-10 production. Using 2-photon microscopy to quantify the entire intra-granuloma B cell population, in conjunction with the adoptive transfer of polyclonal and HEL-specific BCR-transgenic B cells isolated from L. donovani-infected mice, we demonstrated that B cells accumulate in granulomas over time in an antigen-independent manner. Intra-vital dynamic imaging was used to demonstrate that within the polyclonal B cell population obtained from L. donovani-infected mice, the frequency of B cells that made multiple long contacts with endogenous T cells was greater than that observed using HEL-specific B cells obtained from the same inflammatory environment. These data indicate, therefore, that a subset of this polyclonal B cell population is capable of making cognate interactions with T cells within this unique environment, and provide the first insights into the dynamics of B cells within an inflammatory site.

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B cells in granulomas make contacts with endogenous T cells.A. MD-4 and immune B cells were evaluated for contacts with DsRed+ cells in 28 granulomas imaged from two L. donovani-infected Tred mice. The duration of all individual contacts is shown as a separate symbol. B. The frequency of contacts of different duration is shown for MD-4 (open bars) and immune (grey bars) B cells; P<0.001). C. Single frames from Video S1 showing two immune B cells making short (top images) or long (bottom images) contacts with DsRed+ cells. Contact plot is shown below, where each box represents a contact period and adjacent boxes indicate where multiple DsRed+ cells are bound simultaneously. D. The total time each individual B cell (grey bars) spends interacting with any granuloma T cell during the imaging period (i.e. the accumulated total contact time) is shown. Note that due to interactions with multiple T cells, some B cells show an accumulated total contact time longer than the imaging time of 30 min. E. Distribution of contact times for each individual MD-4 (open bars) or immune (grey bars) B cell. Data are expressed as frequency of total incidences for each B cell that involved short (<3 min) or long (>3 min) contact with an endogenous DsRed+ cell. **, p<0.01.
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pone-0034143-g009: B cells in granulomas make contacts with endogenous T cells.A. MD-4 and immune B cells were evaluated for contacts with DsRed+ cells in 28 granulomas imaged from two L. donovani-infected Tred mice. The duration of all individual contacts is shown as a separate symbol. B. The frequency of contacts of different duration is shown for MD-4 (open bars) and immune (grey bars) B cells; P<0.001). C. Single frames from Video S1 showing two immune B cells making short (top images) or long (bottom images) contacts with DsRed+ cells. Contact plot is shown below, where each box represents a contact period and adjacent boxes indicate where multiple DsRed+ cells are bound simultaneously. D. The total time each individual B cell (grey bars) spends interacting with any granuloma T cell during the imaging period (i.e. the accumulated total contact time) is shown. Note that due to interactions with multiple T cells, some B cells show an accumulated total contact time longer than the imaging time of 30 min. E. Distribution of contact times for each individual MD-4 (open bars) or immune (grey bars) B cell. Data are expressed as frequency of total incidences for each B cell that involved short (<3 min) or long (>3 min) contact with an endogenous DsRed+ cell. **, p<0.01.

Mentions: For 134 immune B cells and 121 MD-4 B cells, we determined individual displacement, track length and average velocity and calculated the displacement rate and meandering index (Figure 8A–E). By none of these criteria could we distinguish the behaviour of immune B cells from MD-4 B cells. This result was expected given the highly motile nature of B cell-T cell conjugates seen in lymphoid tissue [17] and the similar degree of mobility of granuloma B cells (Figure 6A) and T cells [33], [35]. We then examined the ability of B cells to engage with endogenous T cells, using the study of Qi et al as a template for our analysis and to allow ready comparison with data obtained using MD-4 B cells in LN [17]. We analysed 1089 individual contacts between MD-4 B cells and endogenous T cells. The average contact duration of 2.81±0.11 min was similar to that observed for non-cognate interactions in the LN follicle. Nevertheless, at a population level, MD-4 B cells made a substantial number of contacts that were longer than expected for non-cognate interactions, mostly between 3 and 10 min of duration but often extending for 30 min (Figure 9A). In contrast to MD-4 B cells, the average contact time for immune B cells was 4.02±0.19 min (n = 756 contacts, P<0.0001; Figure 9A), indicating that at a population level, these immune B cells had sustained engagement with T cells compared to MD-4 B cells. Stratifying the data according to contact time per incidence confirmed that there was a significantly greater trend for polyclonal B cells to make contacts of longer duration than those made by MD-4 B cells (Figure 9B; χ2 = 34.9, df = 4, p<0.0001). We next analysed each individual B cell for the duration of contacts that it made with individual T cells. A range of behaviour was observed, from repeated short interactions to single long contacts that spanned the entire imaging window. Single frame images taken from the imaging videos, and contact plots (where each period of contact is indicated by a white box) are shown for two MD-4 B cells displaying different behaviour during a 30 min imaging window (Figure 9C and Video S1). For each B cell shown, there were also periods when simultaneous contact was made with multiple T cells (shown as adjacent white boxes in Figure 9C). However, we did not observe any B cell division within granulomas during this study. When summated to provide an accumulated total contact time for each B cell, the total contact time for any individual B cell also varied widely (from less than 1 min to over 80 min). It was difficult, however, to discern any significant difference between the behaviour of the immune and MD-4 B cell populations by this approach (Figure 9D). Nevertheless, when we analysed the percentage of short (<3 min) vs. long (3–40 min) contacts made by each B cell population, immune B cells had a significant bias towards making long term contacts compared to the MD-4 B cells (p<0.01; Figure 9E). Although demonstrating a clear difference in behaviour at the population level, due to the variability in behaviour of the MD-4 B cells it was not possible to determine with any degree of statistical certainty the absolute frequency of immune B cells that engaged in these cognate interactions with granuloma T cells.


B cell: T cell interactions occur within hepatic granulomas during experimental visceral leishmaniasis.

Moore JW, Beattie L, Dalton JE, Owens BM, Maroof A, Coles MC, Kaye PM - PLoS ONE (2012)

B cells in granulomas make contacts with endogenous T cells.A. MD-4 and immune B cells were evaluated for contacts with DsRed+ cells in 28 granulomas imaged from two L. donovani-infected Tred mice. The duration of all individual contacts is shown as a separate symbol. B. The frequency of contacts of different duration is shown for MD-4 (open bars) and immune (grey bars) B cells; P<0.001). C. Single frames from Video S1 showing two immune B cells making short (top images) or long (bottom images) contacts with DsRed+ cells. Contact plot is shown below, where each box represents a contact period and adjacent boxes indicate where multiple DsRed+ cells are bound simultaneously. D. The total time each individual B cell (grey bars) spends interacting with any granuloma T cell during the imaging period (i.e. the accumulated total contact time) is shown. Note that due to interactions with multiple T cells, some B cells show an accumulated total contact time longer than the imaging time of 30 min. E. Distribution of contact times for each individual MD-4 (open bars) or immune (grey bars) B cell. Data are expressed as frequency of total incidences for each B cell that involved short (<3 min) or long (>3 min) contact with an endogenous DsRed+ cell. **, p<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316612&req=5

pone-0034143-g009: B cells in granulomas make contacts with endogenous T cells.A. MD-4 and immune B cells were evaluated for contacts with DsRed+ cells in 28 granulomas imaged from two L. donovani-infected Tred mice. The duration of all individual contacts is shown as a separate symbol. B. The frequency of contacts of different duration is shown for MD-4 (open bars) and immune (grey bars) B cells; P<0.001). C. Single frames from Video S1 showing two immune B cells making short (top images) or long (bottom images) contacts with DsRed+ cells. Contact plot is shown below, where each box represents a contact period and adjacent boxes indicate where multiple DsRed+ cells are bound simultaneously. D. The total time each individual B cell (grey bars) spends interacting with any granuloma T cell during the imaging period (i.e. the accumulated total contact time) is shown. Note that due to interactions with multiple T cells, some B cells show an accumulated total contact time longer than the imaging time of 30 min. E. Distribution of contact times for each individual MD-4 (open bars) or immune (grey bars) B cell. Data are expressed as frequency of total incidences for each B cell that involved short (<3 min) or long (>3 min) contact with an endogenous DsRed+ cell. **, p<0.01.
Mentions: For 134 immune B cells and 121 MD-4 B cells, we determined individual displacement, track length and average velocity and calculated the displacement rate and meandering index (Figure 8A–E). By none of these criteria could we distinguish the behaviour of immune B cells from MD-4 B cells. This result was expected given the highly motile nature of B cell-T cell conjugates seen in lymphoid tissue [17] and the similar degree of mobility of granuloma B cells (Figure 6A) and T cells [33], [35]. We then examined the ability of B cells to engage with endogenous T cells, using the study of Qi et al as a template for our analysis and to allow ready comparison with data obtained using MD-4 B cells in LN [17]. We analysed 1089 individual contacts between MD-4 B cells and endogenous T cells. The average contact duration of 2.81±0.11 min was similar to that observed for non-cognate interactions in the LN follicle. Nevertheless, at a population level, MD-4 B cells made a substantial number of contacts that were longer than expected for non-cognate interactions, mostly between 3 and 10 min of duration but often extending for 30 min (Figure 9A). In contrast to MD-4 B cells, the average contact time for immune B cells was 4.02±0.19 min (n = 756 contacts, P<0.0001; Figure 9A), indicating that at a population level, these immune B cells had sustained engagement with T cells compared to MD-4 B cells. Stratifying the data according to contact time per incidence confirmed that there was a significantly greater trend for polyclonal B cells to make contacts of longer duration than those made by MD-4 B cells (Figure 9B; χ2 = 34.9, df = 4, p<0.0001). We next analysed each individual B cell for the duration of contacts that it made with individual T cells. A range of behaviour was observed, from repeated short interactions to single long contacts that spanned the entire imaging window. Single frame images taken from the imaging videos, and contact plots (where each period of contact is indicated by a white box) are shown for two MD-4 B cells displaying different behaviour during a 30 min imaging window (Figure 9C and Video S1). For each B cell shown, there were also periods when simultaneous contact was made with multiple T cells (shown as adjacent white boxes in Figure 9C). However, we did not observe any B cell division within granulomas during this study. When summated to provide an accumulated total contact time for each B cell, the total contact time for any individual B cell also varied widely (from less than 1 min to over 80 min). It was difficult, however, to discern any significant difference between the behaviour of the immune and MD-4 B cell populations by this approach (Figure 9D). Nevertheless, when we analysed the percentage of short (<3 min) vs. long (3–40 min) contacts made by each B cell population, immune B cells had a significant bias towards making long term contacts compared to the MD-4 B cells (p<0.01; Figure 9E). Although demonstrating a clear difference in behaviour at the population level, due to the variability in behaviour of the MD-4 B cells it was not possible to determine with any degree of statistical certainty the absolute frequency of immune B cells that engaged in these cognate interactions with granuloma T cells.

Bottom Line: In infected mice, there was a small increase in mIgM(lo)mIgD(+) mature B2 cells, but no enrichment of B cells with regulatory phenotype or function compared to the naïve hepatic B cell population, as assessed by CD1d and CD5 expression and by IL-10 production.Intra-vital dynamic imaging was used to demonstrate that within the polyclonal B cell population obtained from L. donovani-infected mice, the frequency of B cells that made multiple long contacts with endogenous T cells was greater than that observed using HEL-specific B cells obtained from the same inflammatory environment.These data indicate, therefore, that a subset of this polyclonal B cell population is capable of making cognate interactions with T cells within this unique environment, and provide the first insights into the dynamics of B cells within an inflammatory site.

View Article: PubMed Central - PubMed

Affiliation: Centre for Immunology and Infection, Hull York Medical School and Department of Biology, University of York, Heslington, York, United Kingdom.

ABSTRACT
Hepatic resistance to Leishmania donovani infection in mice is associated with the development of granulomas, in which a variety of lymphoid and non-lymphoid populations accumulate. Although previous studies have identified B cells in hepatic granulomas and functional studies in B cell-deficient mice have suggested a role for B cells in the control of experimental visceral leishmaniasis, little is known about the behaviour of B cells in the granuloma microenvironment. Here, we first compared the hepatic B cell population in infected mice, where ≈60% of B cells are located within granulomas, with that of naïve mice. In infected mice, there was a small increase in mIgM(lo)mIgD(+) mature B2 cells, but no enrichment of B cells with regulatory phenotype or function compared to the naïve hepatic B cell population, as assessed by CD1d and CD5 expression and by IL-10 production. Using 2-photon microscopy to quantify the entire intra-granuloma B cell population, in conjunction with the adoptive transfer of polyclonal and HEL-specific BCR-transgenic B cells isolated from L. donovani-infected mice, we demonstrated that B cells accumulate in granulomas over time in an antigen-independent manner. Intra-vital dynamic imaging was used to demonstrate that within the polyclonal B cell population obtained from L. donovani-infected mice, the frequency of B cells that made multiple long contacts with endogenous T cells was greater than that observed using HEL-specific B cells obtained from the same inflammatory environment. These data indicate, therefore, that a subset of this polyclonal B cell population is capable of making cognate interactions with T cells within this unique environment, and provide the first insights into the dynamics of B cells within an inflammatory site.

Show MeSH
Related in: MedlinePlus