LD of SRBC induces DTH, HD the formation of GC.

<p>Mice were primed with a LD or a HD of SRBC intravenously. 5 days after priming one footpad was challenged with SRBC as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067746#pone.0067746-Hurtrel1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067746#pone.0067746-Lagrange2" target="_blank">[12]</a>. The application scheme is shown in (<b>A</b>). Footpad thickness was measured between 1 and 4 days after challenge (*indicate significant differences in footpad thickness compared to control mice; n = 6–11; data were combined from three independent experiments with 2–5 mice) (<b>B</b>). Cryosections of spleens of LD and HD primed mice were stained immunohistochemically with antibodies against B cells (B220, blue). Proliferating cells were labeled with the proliferation marker Ki-67 (red) (<b>C</b> and <b>D</b>, <b>F</b> and <b>G</b>). Proliferating B cells within GC 10 d after HD priming are shown (<b>C</b>). The area of GC was measured and expressed as percentage of the area of the corresponding B cell follicles (*indicate significant differences between LD and HD primed mice at indicated time points; n = 3–6) (<b>D</b>). Serum from LD and HD primed mice was prepared and SRBC-specific IgGs were measured by ELISA (*indicate significant differences between LD and HD primed mice at indicated time points; n = 3–6) (<b>E</b>). Proliferating cells in spleens primed with a LD or HD and controls are shown (<b>F</b>). Proliferating cells were counted within the TCZ of LD and HD primed mice at indicated time points (*indicate significant differences between the number of proliferating T cells compared to unchallenged mice; n = 3–6) (<b>G</b>). All data are given as mean ± SD (*p<0.05; **p<0.01 Mann-Whitney-U-test). Abbreviations: BCZ, B cell zone; DTH, delayed type hypersensitivity reaction; GC, germinal center; HD, high dose (10⁹)<i>;</i> LD, low dose (10⁵); SRBC, sheep red blood cells.</p

LD Th1 cells appear in the skin, HD Th2 cells in the spleen.

<p>Mice were primed with a LD or a HD of SRBC intravenously. The spleens were snap frozen, sections were prepared and the TCZ and BCZ were isolated by laser-microdissection. To confirm that the TCZ and BCZ are accurately identified and high-quality mRNAs are obtained, we analyzed mRNA expression of <i>Cd3ε</i> and <i>Cd19</i> after laser-microdissection (<b>A</b>). The mRNA expression of the Th1 cytokine <i>Ifnγ,</i> the Th2 cytokine <i>Il4</i> and the marker cytokine for regulatory T cells <i>Il10</i> was analyzed by real-time RT-PCR after isolation of the TCZ of the spleen. Data are means ± SEM. Data are normalized to <i>Mln51</i> mRNA expression levels. Significant differences in the expression of <i>Ifnγ, Il4</i> and <i>Il10</i> between primed mice compared to the controls are shown (n = 6, from two independent experiments with n = 3 mice) (<b>B</b>). 5 days after priming one footpad was challenged with SRBC. Mice were sacrificed 24 and 48 h after challenge and the expression of <i>Ifnγ, Il4</i> and <i>Il10</i> was analyzed in the footpad skins by real-time RT-PCR. Each dot represents the expression level of one mouse. Corresponding means are depicted as black line. Significant differences between LD and HD primed mice are shown (n = 5–11) (<b>C</b>). The mRNA expression of <i>Ifnγ, Il4</i> and <i>Il10</i> was analyzed by real-time RT-PCR after isolation of the BCZ of the spleen, and depicted as described in A (<b>D</b>) (*p<0.05, **p<0.01, Mann-Whitney-U-test). Abbreviations: HD, high dose (10⁹)<i>;</i> LD, low dose (10⁵).</p

Persisting antigen enhances protection.

<p>(A) To establish a potential source of residual antigen (Ag) mice were injected with BCG or Ag85B-TB10.4, 4 weeks (d -28) before irradiation and cell transfer. Control recipients received no source of residual Ag (None) and were irradiated 1 day before cell transfer. CD45RB^low CD4 T cells (3×10⁶) from BCG primed/Ag85B-TB10.4 boosted donors (Pr/Boost, solid symbols) or unprimed CD4 T cells (Naïve, open symbols) were transferred into recipients after irradiation on d 0. All recipients were challenged with BCG 4 weeks later (d 28). (B) Three weeks after challenge spleen cells were assayed for BCG CFUs. Each point represents a single recipient. Horizontal bars are means of 4 recipients/group. ** p<0.01.</p

Protective immunity persists for at least 8 weeks following transfer of prime/boost CD4 T cells.

<p>(A) CD45RB^low CD4 T cells (3×10⁶) from donors primed with BCG and boosted with Ag85B-TB10.4 (Pr/Boost) or 3×10⁶ CD4 T cells from unprimed donors (Naïve) or no cells (None) were transferred to irradiated recipients and challenged with BCG immediately (d 1) or 8 weeks later (d 56) (B, C). Three weeks after challenge spleen cells were assayed for BCG CFUs (B) and ICC IFN-γ⁺ donor CD4 T cells (C). The results are from one experiment. Each point represents a single recipient. Horizontal bars or histograms + SD are means of 4 recipients/group. ** p<0.01.</p

Only lymphocytes of LD primed mice are able to mount a DTH response.

<p>Mice were primed with a LD or a HD of SRBC intravenously. Spleens were removed 5 days after priming; cells were isolated and injected intravenously into naïve recipients. DTH was induced immediately by injection of SRBC into one footpad. The application scheme is shown in (<b>A</b>). Increase in footpad swelling was measured between 1 to 4 days after injection (<b>B</b>). Data are means ± SD. *indicate significant differences in footpad thickness compared to control mice at indicated time points (*p<0.05, **p<0.01; ***p<0.001, Mann-Whitney-U-test, n = 5–8). Abbreviations: DTH, delayed type hypersensitivity reaction; HD, high dose (10⁹)<i>;</i> LD, low dose (10⁵).</p

The development of an adoptive transfer model to evaluate <i>Mtb</i>-specific CD4 T cells.

<p>(A) Donor mice (B6.SJL, CD45.1) injected with BCG 2 mo before were boosted with Ag85B-TB10.4 and killed 7 days later. Spleen and lymph nodes were removed, depleted of B cells, CD8 T cells and macrophages. A representative profile of the resulting CD4 T cell population stained for CD45RB is shown (B). CD4 T cells were also depleted of CD45RB^high cells and injected into recipients on day 0 (d 0). A representative profile of CD45RB^low CD4 T cells used for adoptive transfer is shown (C). One day after transfer, irradiated recipients (X-rad) were challenged i.v. with live BCG, killed 3 weeks later (d 22) and spleens tested for a reduction in BCG numbers (Protection assay, CFUs/spleen). An aliquot of spleen cells was cultured overnight in the presence of Ag85B-TB10.4 antigen and stained for ICC IFN-γ. Donor-derived CD4 T cells (CD4⁺ CD45.1⁺) were gated in R1 (D) and analysed for IFN-γ positive cells (E). The donor-derived CD45.1⁺ CD4⁻ cells in profile D (upper left quadrant) were predominantly contaminating B cells. Numbers in the representative profiles are the percentage of events in the quadrant.</p

Kinetics of the antigen-specific CD4 T cell response in BCG-immunised mice after a boost with Ag85B-TB10.4.

<p>Mice were killed at selected days following antigen injection and spleen cells analysed for ICC IFN-γ. Results are the net percentage of IFN-γ⁺ CD4 T cells stimulated in the presence of Ag85B-TB10.4 after subtraction of the response without antigen. The results were pooled from 8 experiments. Each point represents the mean+SD of 3 or 4 mice, except day 3, n = 2.</p

LTβR dependent growth suppressive activity also affects fully developed splenic tissue.

<p>(A) WT recipients without prior splenectomy were either sham operated or received WT or LTβR^-/- splenic implants. Eight weeks later weight (left side) and cell number (right side) of the endogenous spleen was determined. Only WT splenic regenerates significantly reduced weight and cell number of the endogenous WT spleen. Indicated are means and standard deviation (n = 4–11, * = p < 0.05, ** = p < 0.01). (B) LTβR^-/- recipients without prior splenectomy were either sham operated or received WT or LTβR^-/- splenic implants. Eight weeks later weight (left side) and cell number (right side) of the endogenous spleen was determined. Only WT splenic regenerates significantly reduced weight and cell number of the endogenous LTβR^-/- deficient spleen. Indicated are means and standard deviation (n = 5–8, ** = p < 0.01). These experiments were 2 times independently performed.</p

Identification of LTβR regulated proteins by two-dimensional differential gel electrophoresis and mass spectrometry.

<p>(A) 2D-DIGE experiment performed with splenic stroma of LTβR^-/- (Cy3, green) and WT (Cy5, red) mice. The Cy2 channel is masked (representative gel of three replicas). The encircled area is shown in (B). (B) Gel section showing four spots with an increase in volume ratio when LTβR^-/- (left side) and WT (right side) splenic stroma was compared. Mass spectrometry identified the four spots as four isoforms of MFAP4 (representative gel of three replicas). (C) Indicated is the MFAP4 abundance in splenic stroma of LTβR^-/- spleen, LTβR^-/- spleen from a donor which received a WT splenic implant 8 weeks earlier (LTβR^-/- + WT), and WT spleen. The different 2D-DIGE experiments were compared quantitatively by summing up the volume ratios of the four isoforms of MFAP4 and relating it to the abundance of MFAP4 in LTβR^-/- splenic stroma which was set to one. The abundance of MFAP4 increased from LTβR^-/- splenic stroma to LTβR^-/- splenic stroma obtained from mice with a WT splenic regenerate and further to WT splenic stroma. Indicated are means and standard deviation (n = 3–6). This experiment was 2 times independently performed. (D) Western blot analysis of MFAP4 from splenic stroma lysates of LTβR^-/- mice, LTβR^-/- mice which received a WT splenic implant 8 weeks earlier (LTβR^-/- + WT), and WT mice. Three different sets of experiments are shown.</p

LTβR expression by non-splenic tissues suppresses growth of regenerating splenic tissue.

<p>(A) Macroscopic appearance of splenic regenerates 8 weeks after implantation of wild-type splenic tissue (WT) into wild-type recipients (WT), LTβR deficient splenic tissue (LTβR^-/-) into WT recipients, and WT splenic tissue into LTβR deficient recipients (LTβR^-/-). (B) Weight (left side) and cell number (right side) of splenic regenerates. Indicated are means and standard deviation (n = 4–9, ** = p < 0.01). (C) Microscopic appearance of splenic regenerates. Cryostat sections were stained by immunohistochemistry for T cells (brown; TCRβ⁺) and B cells (blue; B220⁺). Red pulp (RP), T-cell zone (T), and B-cell zone (B) are well developed except in the LTβR^-/- into WT combination where T and B cells are intermixed and only the red pulp (RP) is clearly recognizable (bar: 100 μm). This experiment was 3 times independently performed.</p