Fig 2: DC-intrinsic CARD11-GOF-triggered disease resembles human HLH. (A) The spleen weight was quantified in CARD11GOF-9C and Clec9a-Cre mice (n = 9). (B) Representative consecutive immunohistochemical sections showing the infiltration of macrophages (Iba1) and intrahistiocytic iron (Prussian Blue) in the liver of CARD11GOF-9C mice (n = 4). (C) The aggregation of hemosiderin-laden macrophages was semiquantitatively scored from 0 (absent) to 3 (severe). (D) Histological analysis of HE-stained bone marrow sections revealed hemophagocytosis (shown by arrow) in CARD11GOF-9C mice (n = 1). (E) RBC counts and hemoglobin concentrations were measured by differential hemogram (n = 4). (F–H) The levels of (E) ferritin (n = 11), (F) sCD25 (n = 6), and (G) fibrinogen (n = 4) were quantified by ELISA. (I) The total counts of splenic NK cells were quantified by flow cytometry (n = 6). The frequencies of granzyme B and IFN-γ-producing NK cells in the spleen were analyzed by flow cytometry (n = 3). The gating strategy is shown in SI Appendix, Fig. S2A. The data are presented as the mean ± SD, and unpaired Student’s t test (A, C, and D–I) or two-way ANOVA (I—Granzyme B & IFN-γ) was used to determine statistical significance.

Fig 3: A genetic mouse model for the selective in vivo expression of CARD11-GOF in DCs. (A) The mean CARD11 expression in major splenic immune cell clusters was calculated from the normalized gene expression values using the Tabula Sapiens–Spleen Database (28, 32). (B) The mean Card11 expression in major splenic immune cell clusters was downloaded from the Mouse Cell Atlas 2.0 (29, 33). (C) Schematic illustration of the CARD11GOF x Clec9a-Cre mouse model. (D) Representative flow cytometric plots of the splenic DC subtypes CD8a+ cDC1 and CD11b+ cDC2 and their eGFP expression are shown for CARD11GOF-9C and Clec9a-Cre mice. DC subtypes were pregated on CD11c+ MHC II+ DCs as shown in SI Appendix, Fig. S1C. (E) The frequencies of eGFP in major splenic immune cell populations were quantified by flow cytometry (n = 9). The representative gating strategy for immune cell populations and eGFP-expressing cells is shown in SI Appendix, Fig. S1 C and D. The data are presented as the mean ± SD.

Fig 4: KIN-8194 overcomes secondary resistance to BTKi.A Number of viable JeKo-1 or JeKo-R cells following 7 days of treatment with ibrutinib, acalabrutinib, or KIN-8194. B Number of viable JeKo-1, Mino, and Maver-1 cells transduced with an LZRS-GFP empty vector (EV) control or LZRS-BTKC481S-GFP following 7 days of treatment with various concentrations of ibrutinib, acalabrutinib, or KIN-8194. C Schematic representation of CARD11 domains and the amino acids deleted in Mino-Cas9CARD11-Δ350-355. D Western blot analysis of Mino cells expressing Cas9 (Mino-Cas9) and Mino-Cas9CARD11-Δ350-355 (Mino Δ350–355) cells treated for 6 h (P-BTK, BTK, top tubulin) or 24 h (CYLD cleavage, bottom tubulin) with 100 nM ibrutinib, acalabrutinib, or KIN-8194. cl-CYLD indicates MALT1-mediated cleaved CYLD fragments of 70 and 40 kDa. Tubulin serves as a loading control. E Number of viable Mino-Cas9 or Mino-Cas9CARD11-Δ350-355 cells following 7 days of treatment with ibrutinib, acalabrutinib, or KIN-8194. The number of viable cells was determined by flow cytometric analysis and 7-AAD staining. The number of viable cells was normalized to the DMSO-treated condition. Data are presented as mean ± S.E.M. of at least three independent experiments performed in triplicate. F Western blot analysis of Mino cells and Mino cells transduced with pMSCV-CARD11D357E treated for 6 h (P-BTK, BTK, top tubulin) or 24 h (CYLD cleavage, CARD11, bottom tubulin) with 100 nM ibrutinib, acalabrutinib, or KIN-8194. cl-CYLD indicates MALT1-mediated cleaved CYLD fragments of 70 and 40 kDa. Tubulin serves as a loading control. G Number of viable Mino cells or Mino cells transduced with pMSCV-CARD11D357E following 7 days of treatment with ibrutinib, acalabrutinib, or KIN-8194. The number of viable cells was determined by flow cytometric analysis and 7-AAD staining. The number of viable cells were normalized to the DMSO-treated condition. Data are presented as mean ± S.E.M. of at least three independent experiments performed in triplicate.

Fig 5: Identification of CARMA1 as a binding partner of CKIP-1.(A) HEK293T cells were transfected with plasmids encoding CKIP-1 together with Myc-CARMA1 or PKCθ, lysed, and immunoprecipitated by anti-CKIP-1 or control antibody. (B) HEK293T cells were co-transfected with plasmids encoding Myc-CARMA1, PKCθ or CKIP-1, lysed, and immunoprecipitated by anti-Myc antibodies. (C) HEK293T cells were transfected with DsRed-CKIP-1. 24 hr later, the transfected cells were incubated with Alexa Flour 488-conjugated cholera toxin B (CTx), and were fixed and stained with DAPI. In lower panels, HEK293T cells were transfected with DsRed-CKIP-1 together with EGFP-CARMA1. 24 hr later, cells were fixed and stained with DAPI. The localization of CKIP-1, CARMA1 and Alexa Flour 488-CTx-labeled lipid rafts was visualized by confocal microscopy. (D) JPM50.6/WT cells, which were reconstituted by Myc-CARMA1 WT into CARMA1-deficient Jurkat T cells, were lysed and immunoprecipitated by anti-CKIP-1 or control antibody. Ig hc, immunoglobulin heavy chain. (E) Schematic diagram of CARMA1 and CKIP-1 truncated forms used in the experiments. CARD, caspase recruitment domain; CC, coiled-coil; SH3, Src homology 3; GUK, guanylate kinase; MAGUK, membrane-associated GUK; PH, pleckstrin homology; LZ, leucin zipper. (F) HEK293T cells were transfected with CKIP-1 together with Myc-CARMA1 truncated form, lysed, and immunoprecipitated by anti-Myc antibody, followed by Western blotting with indicated antibodies. (G) HEK293T cells were transfected with Myc-CARMA1 together with each CKIP-1 truncated form. Cell lysates were immunoprecipitated by anti-Myc antibody, followed by Western blotting with indicated antibodies. (H) CARMA1 CD-CC was purified from E. coli as a GST fusion protein. GST alone or GST-tagged CARMA1 CD-CC was incubated with in vitro transcribed/translated FLAG-CKIP-1. GST pull-downs and input were subjected to Western blotting with anti-FLAG antibody.