<b>Mobilization of T cells is strain and TLR2-dependent.</b>

<p> F1 (SJLxB6^wt), F1 (SJLxB6^tlr−) (<b>A</b>, <b>C</b>) or SJL (B, <b>D</b>) mice were immunized with p139 in IFA in the presence of the indicated amounts of killed <i>M tb</i> (<b>A</b>), or of PPD (<b>B</b>, <b>C</b>) or of a 1∶1 w/w mixture of PAM2-(CSK)3 and PAM3-(CSK)3 (<b>D</b>). The number of mice for each group is indicated in the figure. Fourteen days later mice were sacrificed and the presence of T cells carrying the public TCR-beta chain in LN (closed symbols) and spleen (open symbols) was measured by immunoscope. Data are reported as RSI for the peak corresponding to the public BV10 TCR-beta chain for each individual mouse. Dashed lines indicate the cut off value for positivity (established as described in Results). <b>E</b>: 4 F1 (SJLxB6^wt, circles) mice and 5 F1 (SJLxB6^tlr2−, triangles) mice were immunized with regular CFA in PBS. Two weeks later cells from draining LN (full symbols) and spleen (open symbols) were recovered, labeled with CFSE and cultured in the presence or absence (background) of PPD. After 3 days, the number of CD3⁺ cells that had specifically divided in response to PPD was determined as % of CFSE^low CD3⁺ cells over total CD3⁺ cells in the PPD stimulated sample minus % of CFSE^low CD3⁺ cells over total CD3⁺ cells in background sample.</p

<b>Amount of M tuberculosis in the adjuvant modulates appearance of p139-specific-T cells in the SJL strain.</b>

<p>SJL mice were immunized with p139 in IFA containing or not 50 or 200 microgrammes/mouse of <i>M tuberculosis</i> (regular or enriched CFA, respectively). In all the figures, closed symbols refer to LN cells and open symbols to spleen cells. <b>A</b>) Time course of appearance of p139-specific BV10⁺ cells in LN and spleen following challenge with antigen in regular CFA. BV10⁺, p139-specific T cells were measured by immunoscope in draining LN and spleen. <b>B</b>) Presence of p139-specific BV10⁺ cells in the spleen at d 14 after s.c. immunization depends on the amount of M tuberculosis in the adjuvant. SJL mice were immunized s.c. with 100 microliters of a 1∶1 suspension of p139 in regular CFA (11 mice), in enriched CFA (6 mice) or in IFA alone (8 mice). Two weeks later, mice were sacrificed and LN and spleen were examined for the presence of p139-specific BV10⁺ cells by immunoscope. Data are reported as R.S.I., and each symbol represents LN or spleen of one mouse, and the dashed line represents the cut off value for positivity in SJL mice. <b>c</b>) The number of p139 specific T cells in the spleen 14 d after challenge with peptide in enriched CFA is inversely related to the number of the same cells in the LN. SJL mice were immunized s.c. with p139 in enriched CFA (4 mice). Two weeks later, cells from draining LN and spleen were stained with CFSE and cultured in the presence or absence of 10 microgrammes/ml of p139. After 3 days, cells were recovered and stained with PE-labelled anti CD4 monoclonal antibody. p139-specific cells are calculated as CFSE^low CD4⁺ cells in the ag-stimulated sample minus the number of the same cells in the non-stimulated sample.</p

<b>Identification of polymorphisms of TLR2 between SJL and B6 strains and expression of TLR2 on immune cells.</b>

<p><b>A</b>) Comparison of the sequence of TLR2 of SJL and B6. The non-synonymous and synonymous polymorphisms are boxed. Base (first line) and aminoacid (second line) sequences of B6 and the corresponding sequences (third and fourth lines, respectively) of SJL around the polymorphic residues are reported. <b>B</b>) Identification of enriched activated T cells, naïve T cells and APC in LN cells by scattering properties. SJL, B6^wt, F1 (SJLxB6^wt) and F1 (SJLxB6^tlr2−) mice were immunized with IFA containing 50 microgrammes/mouse of <i>M tb</i>.. 8 days later, mice were sacrificed and cells from draining LN were loaded with CFSE and stimulated with PPD. After 3 days, cells were collected and stained with PE-labeled anti-CD3 monoclonal antibody. The colours define cells examined for TLR2 expression in panel C. <b>C</b>) SJL, B6^wt, F1 (SJLxB6^wt) and F1 (SJLxB6^tlr2−) mice were immunized with IFA containing 50 microgrammes/mouse of <i>M tb</i>. Eight d later, mice were sacrificed and cells from draining LN were stimulated with PPD. After 3 days, cells were collected and stained with PE-labeled anti-CD3 mAb and with FITC labeled anti-TLR2 mAb. Expression of TLR2 was evaluated on high scattering CD3⁺ cells (activated T cells, red line), low scattering CD3⁺ cells (naïve T cells, shaded area) and CD3⁻ cells (mostly APC, black line), as shown in <b>B</b>.</p

<b>Appearance of T cells in the spleen is regulated by TLR2 expressed on T cells.</b>

<p><b>A</b>Detection of p139-specific T cells in SJL^BV10 mice. One SJL^BV10 mouse (at the 6^th backcross onto SJL background) was immunized s. c. with p139 in regular CFA, as described above. Ten days later LN cells were obtained, stained with CFSE and cultured in the presence or absence of p139. The number of BV10⁺, p139-specific T cells in the draining LN is measured as the number of CFSE^low BV10⁺ cells in the ag-stimulated sample, minus the number of the same cells in the non-stimulated sample. <b>B</b>) T cells were enriched from the spleen of naïve F1 mice of (SJL^BV10×B6^wt) or (SJL^BV10×B6^tlr2−), and transferred i. p. into naïve F1 mice of (SJL×B6^wt) or (SJL×B6^tlr2−). A week later, recipient mice were immunized s. c. with p139 in regular CFA, and after, further 14 days, cells from LN and spleen were prepared, and the presence of BV10⁺, p139-specific T cells was measured as described in Fig. 4a. The 11 non-transferred mice were 6 F1 (SJL×B6^wt) mice and 5 F1 (SJL×B6^tlr2−) mice, all challenged with p139 in regular CFA. Data are expressed as the number of BV10⁺, CFSE^low, p139-specific cells/10⁴ BV10⁺ cells.</p