Supplemental Information. Inflammatory Ly6C high Monocytes Protect. against Candidiasis through IL-15-Driven. NK Cell/Neutrophil Activation

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1 Immunity, Volume 46 Supplemental Information Inflammatory Ly6C high Monocytes Protect against Candidiasis through IL-15-Driven NK Cell/Neutrophil Activation Jorge Domínguez-Andrés, Lidia Feo-Lucas, María Minguito de la Escalera, Leticia González, María López-Bravo, and Carlos Ardavín

2 !"#$%&,-./ '()*+,-0234,-2534, "78%9+34:&;<,-0 :&;,,-;. :&;,?? =>,?? :&;, C" 4 E*$*9&)%D MK/L "FE*-,D 5K.L E*-,D 0KNL A%D"#%$)4=HD.5K@L???,-.21 =>,?? 9-,D.K5L :&;, 8*G 4 DB8%%$,-;. A%D"#%$)4=HD 5KOL,-0,-0 $%()A*BC"8D 0K0L :&;,,-;. :&;,? =>,?? :&;, C" 4 E*$*9&)%D 5K;L "FE*-,D 5K0L E*-,D 5K0L?? 9-,D 0KML??? :&;, 8*G 4 E*$*9&)%D 5K@L,-.21 =>,??,-0,-0234,-2534, "78%9+34:&;<,-@,-009,-009 Figure S1. Characterization by flow cytometry of DC, monocyte, MØ, NK cell and neutrophil subsets in the kidney and spleen of C57BL/6 mice. Related to Figures 1 and 2.

3 A B WT C D Ccr2- /- Figure S2. Histopathology of the C. albicans-infected kidneys of WT and Ccr2-/mice. Related to Figure 3. (A) Representative PAS-stained histological section from a paraffin-embedded WT mouse kidney at 48 hours after C. albicans infection. (B) High-magnification of the area marked by a blue square in (A). (C) Representative PAS-stained histological section from a paraffin-embedded Ccr2-/- mouse kidney at 48 hours after C. albicans infection. (D) High-magnification of the area marked by a blue square in (C). Scale bars: 500 µm (A and C), 100 µm (B and D).

4 * 5 +,", -.'/.0'/ +,", -.'/.0'/ +,", -.'/.0'/ -3 -)3.3!(12* )3 4%5!(12* Figure S3. Analysis of kidney NK cell activation after systemic C. albicans infection. Related to Figure 3. (A-B) IFNγ and Granzyme B (GrB) production by kidney NK cells analyzed by flow cytometry after intracellular staining in WT (A) and Ccr2 -/- (B) mice at the indicated times after C. albicans infection. Data are expressed as mean ± SEM of four mice per condition. ***p<0.0; unpaired t test. Red asterisks indicate the statistical significance of the differences observed between WT and Ccr2 -/- mice. Similar results were obtained in 3 independent experiments.

5 Figure S4. Fungal burden in the kidney and spleen after systemic C. albicans infection. Related to Figure 4. Kidney and spleen fungal burden of C57BL/6 at the indicated times after C. albicans infection. Data are expressed as mean ± SEM of four mice per condition. *p<0.05; **p<0.; ***p<0.0; unpaired t test. Similar results were obtained in 2 independent experiments.

6 2 4567(!'+&8&+. '.:+&&;'7<==* > '?&++. > '?&++. *+,,-'.&%$/ "#$% &'& 3 ()*+'&9:%&..",; 7,-./0/-!@"++";# '.:+&&;'7<==* > '?&++. '@"-;&A'7<B) > '?&++. ';&$G%,:H"+. "#$% &'& "#$% &'& < C"-;&A'D$;#E+'+,E- F ($%8"8E+!,-./0/-!@"++";# '/,;,?AG&. "#$% &'& "#$% &'& Figure S5. Effect of IL-15 deficiency in defense against C. albicans infection. Related to Figure 4. (A) GM-CSF analyzed by real-time PCR normalized to β-actin in CD11b + spleen cells and CD45 + kidney infiltrating leukocytes, and by ELISA in blood serum, from WT and Il15 -/- mice at the indicated times. Real-time PCR data are expressed as fold induction relative to uninfected mice. (B) Expression of mrna for inos by CD11b + spleen cells and CD45 + kidney infiltrating leukocytes from WT and Il15 -/- mice analyzed by real-time PCR, normalized to β-actin, at the indicated times. Real-time PCR data are expressed as fold induction relative to uninfected mice. (C) Candidacidal activity of blood neutrophils from WT and Il15 -/- mice. (D) Kidney fungal burden of WT mice and Il15 -/- mice at 72 h post-infection. (E) Survival of WT mice and Il15 -/- mice post-infection. n=12. (F) Candidacidal activity of bone marrow Ly6C high monocytes from WT and Il15 -/- mice. Data are expressed as mean ± SEM of 4 mice per condition. *p<0.05; **p<0.; ***p<0.0; unpaired t test. Similar results were obtained in 2 independent experiments. ELISA data for non-infected mice were below the detection level of the ELISA kit (63 pg/ml). n.i., non-infected; n.d., not detectable.

7 4 67-2(''% )*+* $,-. +%/&(-2/)03/' +%/&(-2/)03/' %&'(() 5 )(803/&.+'% )*+* $,-. +%/&(-2/)03/' +%/&(-2/)03/' %&'(() Figure S6. CD122 expression by spleen NK cells and neutrophils. Related to Figure 5. CD122 expression by spleen NK cells and neutrophils of non-infected C57BL/6 mice and C57BL/6 mice 24 hr after infection. Data are expressed as mean fluorescence intensity (MFI) ± SEM of four mice per condition. ***p<0.0; unpaired t test.

8 * -./0) -./0) 1 +, +,!"#$%& '(' )*+, '(' Figure S7. Dectin-1 and IRF5 deficiency are related with an impaired IL-15 expression. Related to Figure 6. (A and B) Expression of mrna for IL-15 by BMDCs from WT and Clec7a -/- (A) or Irf5 -/- (B) mice analyzed by real-time PCR, normalized to β-actin, 6 hours after stimulation with HKC, Curdlan or LPS. Data are expressed as fold induction relative to unstimulated cells. Data represent mean ± SEM of four independent BMDC cultures per condition. *p<0.05; **p<0.; ***p<0.0; unpaired t test. Similar results were obtained in 2 independent experiments.

9 Table S1. Primers for Real-Time PCR. Related to the STAR Methods section. Gene Primer Sequence 5-3 Il2 Il12A Il12B Il15 Il18 Il23A Csf2 Nos2 b-actin Il2-for CAAGCAGGCCACAGAATTGA Il2-rev CCGCAGAGGTCCAAGTTCA Il12A-for CCACCCTTGCCCTCCTAAA Il12A-rev GGCAGCTCCCTCTTGTTGTG Il12B-for GGAAGCACGGCAGCAGAATA Il12B-rev AACTTGAGGGAGAAGTAGGAATGG Il15-for CATCCATCTCGTGCTACTTGTGTT Il15-rev CATCTATCCAGTTGGCCTCTGT Il18-for CAGGCCTGACATCTTCTGCAA Il18-rev CTGACATGGCAGCCATTGT Il23A-for GCTGTGCCTAGGAGTAGCAG Il23A-rev CACTGGATACGGGGCACATT Csf2-for GGTCCTGAGGAGGATGTG Csf2-rev GAGGTTCAGGGCTTCTTTGA Nos2-for CAGCTGGGCTGTACAAACCTT Nos2-rev CATTGGAAGTGAAGCGTTTCG b-actin-for GGCTGTATTCCCCTCCATCG b-actin-rev CCAGTTGGTAACAATGCCATGT