Haasken et al.

Macrophage scavenger receptor 1 (Msr1, SR-A) influences B cell autoimmunity by regulating soluble autoantigen concentration

Supplemental Figure 1: Decreased incidence and severity of arthritis and endocarditis in

Msr1–/– K/BxN mice. (A) Histological analysis of ankle (left panels) and mitral valve (right panels) pathology in Msr1+/+, +/–, and –/– K/BxN mice demonstrates protection from both joint and cardiac valve pathology in non-arthritic Msr1–/– K/BxN mice. For ankles, original magnification = 10x, bar (in fourth row) indicates 200 microns. For mitral valves, original magnification = 40x; bar (in fourth row) indicates 100 microns. (B) Five non-arthritic Msr1–/–

K/BxN mice (filled circles) were aged up to 15 weeks, during which any mild arthritis resolved or remained equivalent to the 8-week time point, as demonstrated by changes in ankle thickness.

The data from the Msr1+/+ K/BxN mice (reproduced from Figure 1B) are shown for comparison.

Plotted values are means ± SEM. *p 0.05, **p 0.01.

Supplemental Figure 2: Msr1-sufficient host environment permits Msr1-deficient K/BxN hematopoietic compartment to drive arthritis and B cell activation

Rag1–/– recipient mice were sublethally irradiated (300 Rad) and transplanted with 10x106 bone marrow cells from Msr1+/+ K/BxN mice or Msr1–/– K/BxN mice as indicated. (A) The development of arthritis in both groups was determined by weekly arthritis scoring and ankle measurements. Values shown are means +/- SEM. n= 3 mice/group. (B) B cells in the reconstituted mice were analyzed for their ability to bind to GPI-tetramer, for having undergone isotype switching express IgG1 intracellularly, and transitioning to a CD38low GL7+ activated phenotype. Numbers indicate the percentage of cells in each quadrant or gate. (C)

Representative flow cytometric plot of CD3– F4/80+ in the reconstituted mice analyzed for expression of the congenic markers differentiating the donor cells (CD45.1+

CD45.2+) from the host cells (CD45.1– CD45.2+). The numbers shown are percentages. In analysis of multiple mice, the percent of macrophages that were host-derived ranged from 38-

63%. (D) Similar analysis for CD3+ T cells and B220+ B cells reveals that they are entirely donor-derived. Haasken et al. Supplemental Figure 1

A ankle mitral valve

KRN negative

Msr1 +/+ K/BxN

Msr1 +/– K/BxN

Msr1–/– K/BxN (arthritic)

Msr1–/– K/BxN (non-arthritic)

B 1.8 ** ** K/BxN 1.6 ** Msr1+/+ 1.4 Msr1–/– * 1.2 1.0 0.8 0.6 Ankle Width (mm) Ankle Width 0.4 Δ 0.2 0 3691215 Age of mice (weeks) Haasken et al. Supplmental Figure 2

A 12 donor marrow 1.30 10 Msr1+/+ K/BxN 1.10 Msr1–/– K/BxN 8 0.90 6 0.70 0.50 4 Arthritis Score 0.30 2 (mm) Ankle Width Δ 0.10 0 0123456 0123456 Time after transplantation (weeks) Time after transplantation (weeks) B gated B220+ gated GPI+ Gate 1 Gate 2 donor 62 0.42 7.02 0.21 G1 marrow 8.93 Msr1 G2 +/+ G2 K/BxN 0.19 88.7

35.3 2.3 86.9 5.8

59.6 5.66 5.75 0.7 G1 16.5 Msr1 G2 –/– G2 K/BxN 0.13 78.2

decoy tetramer IgM CD38 31.4 3.46 CD38 81.1 12.5 GPI tetramer intracell. IgG1 GL7

CD donor 99.9

CD45.1 0.042 donor host 42.1

CD45.2 CD3 CD3

CD45.1 54.8 host donor 99.7 F4/80 CD45.2 B220

CD45.1 0.21 host

CD45.2