Cantin, Edouard; JVI01648-07
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Appendix.
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Supplementary Figure Legends.
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Supplemental Figure 1.
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reactivity between CD11b, CD11c and Gr-1 in cells from 129S6 mice.
Effects of depletion treatments and determination of cross-
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20,000-50,000 events are shown. Intraperitoneal administration of protein G purified
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anti-Gr-1 mAb depletes mature Gr-1+CD11b+ two days after last injection as 6.1% of
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splenocytes are double-positive in PBS control mice (A) versus 0.4% of splenocytes from treated
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mice (B). Correspondingly, the values are reversed when quantitating re-emerging (CD11b+Gr-
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1Lo/Int) cells (0.7% and 6.6%, respectively).
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Multiple populations among CD11c+ (C) and CD11b+ (H) cells cross-react with Gr-1.
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Five different sub-populations of CD11b and CD11c stained splenocytes (F) were examined for
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Gr-1 positivity (D, E, G, I, J). The greatest proportion of Gr-1+ cells (74%) are found in
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CD11b+CD11c- cells whereas 25% of CD11b+CD11c+ DCs also stain for Gr-1.
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CD11b-CD11c- cells only harbor 1.8% Gr-1+ cells, the absolute number for these Gr-1+ cells is
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close to half of the CD11b+CD11c+Gr-1+ number.
Although
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Depletion with clodronate and/or Gr-1 mAb does not affect CD4+ T cells or DX5+ (pan-
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NK) cells (K) whereas a slight drop in CD8+ occurs when mice are treated with clodronate
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(possibly due to elimination of CD8+ DCs) and a reduction by close to 50% is seen in
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splenocytes from mice treated with Gr-1 mAb alone or in combination with clodronate (L).
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Cantin, Edouard; JVI01648-07
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Peritoneal exudate cells (PECs) were used to determine the efficacy of clodronate to
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deplete CD11b+ cells. Two days after the last intraperitoneal injection, PECs were collected by
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lavage and stained for CD11b and CD80. Greater than 95% depletion of CD11b+ cells occur
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after treatment (about 9500 events for PBS control, M, compared to 450 for clodronate treated
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mice, N) and, in addition, about 2/3 of remaining cells (N) show an activated phenotype (CD80+)
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when only about 1/6 of control mice are CD80+ (M).
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Supplemental Figure 2.
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mice.
Comparison of splenocyte populations from B6 and B6- B6-E
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Single cell preparations from 2 mice per strain were stained for combinations of various
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cell surface markers. Data shown are representative of two separate analyses. It should be noted
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that total cell recovery from B6 mice are 3.5-6.0 times greater than from B6-E mice. In all
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panels, 20,000-50,000 events are shown.
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(A),
Slight changes in cell populations in B-6E compared to B6 mice. FSC-SSC plots show
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relative increase of larger and more granular cells in B6-E mice. CD19+ early pro-B cells are
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present in B6-E (4.7%) while in C57BL/6 the same gate (53%) includes all stages of immature
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and mature B-cells. A small proportion of CD4+ cells (1.3%) remain in B6-E, about a quarter of
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which express CD11c (see B. below), compared to normal levels (16.5%) of CD4+ cells (mostly
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T lymphocytes) in B6. Further characterization of these CD4+ cells was not done. It is notable
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that the absolute numbers of NK1.1+ cells was unchanged between B6-E (19.7%) and B6
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(5.2%), their relative ratio being 19.7/5.2=3.8. CD8+ cells still remaining in B6-E are primarily
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CD11c+CD8+ DCs (see B. below), and although Gr-1HI and Gr-1INT cells are more prevalent in
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Cantin, Edouard; JVI01648-07
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B6-E, their absolute numbers are relatively unchanged since the recovery of cells in B6 is
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greater. This is also true for the various sub-populations of F4/80 vs CD11b and Gr-1 vs CD11b
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stained cells (relative ratios 3.0-6.9) with the exception of CD11b+F4/80- cells (B6E:B6 ratio =
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10.8, a 2-fold increase in B6-E) and CD11b+Gr-1Lo cells (ratio 1.3, 3-fold reduction in B6-E).
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The consequences of these shifts in cell populations remain unclear and their investigation is
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beyond the scope of this study.
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(B),
CD4 and CD8 positive dendritic cells in B6-E mice. The majority of “larger” CD8+
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cells (1.39% in B6-e and 1.79% in B6) in B6-E are CD11c+ (71%) indicating that they are DCs
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while, proportionally, the “smaller” CD8+ (0.94% in B6-E and 4.75% in B6) and the CD4+
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(2.93% in B6-E and 13.7% in B6) cells are equal in the two strains (CD11c expression around
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23% in B6-E and 2.3% in B6, respectively). Since B6 mice have CD8+ and CD4+ T cells, a
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direct comparison of absolute numbers in this analysis is not possible without additional
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antibody staining combinations.
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MHCII expression in Ly-6C+ (pre-gated) plasmacytoid and non-plasmacytoid dendritic
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(C),
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cells. The relative number of PDCA-1+ cells in B6-E mice is approximately the same as in B6
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mice although a greater proportion are CD11c+ (57% vs 35% for B6). Absolute numbers of DCs
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in B6-E mice are 2-3 fold lower than in B6 and MHCII expression in PDCA-1+ cells is about 2-
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fold lower in B6-E mice.. Approximately 60% of non-plasmacytoid (PDCA-1- CD11c+) DC in
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both B6-E and B6 mice were MHCII+.
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