Type I IFN Is Necessary and Sufficient for Inflammation-Induced Red Blood Cell Alloimmunization in Mice

This information is current as David R. Gibb, Jingchun Liu, Prabitha Natarajan, Manjula of September 23, 2021. Santhanakrishnan, David J. Madrid, Stephanie C. Eisenbarth, James C. Zimring, Akiko Iwasaki and Jeanne E. Hendrickson J Immunol 2017; 199:1041-1050; Prepublished online 19

June 2017; Downloaded from doi: 10.4049/jimmunol.1700401 http://www.jimmunol.org/content/199/3/1041

Supplementary http://www.jimmunol.org/content/suppl/2017/06/17/jimmunol.170040 http://www.jimmunol.org/ Material 1.DCSupplemental References This article cites 67 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/199/3/1041.full#ref-list-1

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Type I IFN Is Necessary and Sufficient for Inflammation-Induced Red Blood Cell Alloimmunization in Mice

David R. Gibb,* Jingchun Liu,* Prabitha Natarajan,* Manjula Santhanakrishnan,* David J. Madrid,† Stephanie C. Eisenbarth,*,‡ James C. Zimring,x,{,‖ Akiko Iwasaki,‡,# and Jeanne E. Hendrickson*,†

During RBC transfusion, production of alloantibodies against RBC non-ABO Ags can cause hemolytic transfusion reactions and limit availability of compatible blood products, resulting in anemia-associated morbidity and mortality. Multiple studies have established that certain inflammatory disorders and inflammatory stimuli promote alloimmune responses to RBC Ags. However, the molecular mechanisms underlying these findings are poorly understood. Type I IFNs (IFN-a/b) are induced in inflammatory conditions associated with increased alloimmunization. By developing a new transgenic murine model, we demonstrate that signaling through the IFN-a/b receptor is required for inflammation-induced alloimmunization. Additionally, mitochondrial Downloaded from antiviral signaling –mediated signaling through cytosolic pattern recognition receptors was required for polyinosinic- polycytidylic acid–induced IFN-a/b production and alloimmunization. We further report that IFN-a, in the absence of an adjuvant, is sufficient to induce RBC alloimmunization. These findings raise the possibility that patients with IFN-a/b–mediated conditions, including autoimmunity and viral infections, may have an increased risk of RBC alloimmunization and may benefit from personalized transfusion protocols and/or targeted therapies. The Journal of Immunology, 2017, 199: 1041–1050. http://www.jimmunol.org/

uring allogeneic RBC transfusion, most non-ABO Ags on recipients and as many as 30–50% of transfusion-dependent pa- RBCs are not routinely matched between donor units and tients with sickle cell disease (1–7). Such responses can cause D recipients. Hence, transfusion exposes recipients to nu- potentially fatal hemolytic transfusion reactions (8, 9). Moreover, merous RBC alloantigens, including Kell, Duffy, and Kidd, which substantial morbidity and mortality can occur for patients who induce formation of Ag-specific IgG alloantibodies in 3–10% of all have alloantibodies against multiple Ags. Units of compatible RBCs are more difficult to locate, resulting in delays in therapy, and in extreme cases, death from lack of compatible blood (10,

*Department of Laboratory Medicine, Yale University School of Medicine, New 11). However, the majority of transfusion recipients do not form by guest on September 23, 2021 † Haven, CT 06520; Department of Pediatrics, Yale University School of Medicine, alloantibodies and tools allowing prediction of such responses are New Haven, CT 06520; ‡Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520; xBloodworks Northwest Research Institute, Seat- not currently available. Thus, characterization of factors that { tle, WA 98102; Department of Laboratory Medicine, University of Washington promote RBC alloantibody responses could allow for identifica- School of Medicine, Seattle, WA 98195; ‖Division of Hematology, Department of Medicine, University of Washington School of Medicine, Seattle, WA 98195; and tion of at-risk patients and intervention to mitigate alloimmuni- #Howard Hughes Medical Institute, Chevy Chase, MD 20815 zation and its detrimental effects. ORCIDs: 0000-0003-3703-7635 (M.S.); 0000-0002-1244-208X (S.C.E.); 0000-0002- Recent human studies have confirmed earlier mouse experiments 7824-9856 (A.I.); 0000-0002-7928-3132 (J.E.H.). indicating that the inflammatory state of transfusion recipients can Received for publication March 17, 2017. Accepted for publication May 23, 2017. influence the frequency of RBC alloantibody responses. Elevated This work was supported by grants from the National Blood Foundation (R13672) (to alloimmunization rates have been reported for patients with acute D.R.G.) and the National Institutes of Health/National Heart, Lung, and Blood In- chest syndrome, febrile transfusion reactions, and autoimmune stitute (R01 HL126076) (to J.E.H.) and (T32 HL007974-14) (to Brian Smith, Chair of the Department of Laboratory Medicine, Yale University School of Medicine). diseases, including systemic lupus erythematosus (SLE) and in- D.R.G., A.I., J.C.Z., S.C.E., and J.E.H. planned the experiments completed by D.R.G., flammatory bowel disease (12–15). Also, similar to prior murine J.L., P.N., M.S., D.J.M., and J.E.H; J.C.Z. generated K1 mice. All authors made studies, a recent human study reported that inflammation associ- experimental suggestions. D.R.G. wrote the initial draft of the manuscript, and all authors edited the manuscript and approved the final version. ated with different infections can have distinct effects. Patients with viremia were reported to have increased rates of alloimmu- This work was presented in abstract form at the annual meeting of the American Society of Hematology, December 3, 2016, San Diego, CA. nization, whereas patients with Gram-negative bacterial infections Address correspondence and reprint requests to Dr. Jeanne E. Hendrickson, Yale had lower rates (16). These associations indicate that specific University Departments of Laboratory Medicine and Pediatrics, 330 Cedar Street, pathways activated in some inflammatory conditions, such as Clinic Building 405, PO Box 208035, New Haven, CT 06520. E-mail address: [email protected] autoimmunity and viral infections, promote RBC alloimmunization. However, examination of these pathways, including inflammatory The online version of this article contains supplemental material. signaling, has only just begun (17, 18). Abbreviations used in this article: cDC, conventional DC; DC, dendritic cell; HEL, hen egg lysozyme; IFNAR1, IFN-a and -b receptor 1; IRF, IFN regulatory factor; Given the large number of antigenic differences between human MAVS, mitochondrial antiviral signaling protein; MDA5, melanoma differentiation– donors and recipients, multiple groups have used murine trans- associated 5; MHCII, MHC class II; pDC, plasmacytoid DC; poly(I:C), polyinosinic-polycytidylic acid; rIFN-a, recombinant IFN-a; RIG-I, retinoic acid fusions models that allow examination of alloimmune responses to inducible gene-I; RLR, retinoic acid inducible gene-I–like receptor; SLE, systemic a single donor RBC Ag (19–21). In the absence of an adjuvant, lupus erythematosus; TRIF, TIR-domain–containing adaptor protein inducing IFN-b; transfusion of mouse RBCs expressing human or model RBC Ags WT, wild type; YFP, yellow fluorescent protein. results in low-level alloimmune responses in some donor models and Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 no response in others. However, treatment of transfusion recipients www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700401 1042 IFN-a/b REGULATES RBC ALLOIMMUNIZATION with inflammatory pathogen-associated molecular patterns has been important regulator of antiviral immunity and autoimmune pa- shown to induce or enhance RBC alloimmune responses (22). thology. IFN-a/b includes a single IFN-b and 13 IFN-a Cotransfusion with CpG DNA or pretreatment with polyinosinic- that signal through a ubiquitously expressed dimeric receptor, polycytidylic acid [poly(I:C)], a mimetic of viral dsRNA, was shown consisting of IFN-a and -b receptor 1 (IFNAR1) and IFNAR2. to induce alloimmunization to human glycophorin A expressed on Signal transduction results in the expression of numerous IFN- mouse RBCs (21, 23). In addition, poly(I:C) has been shown to en- stimulated that inhibit viral replication and dissemination hance the magnitude of alloimmune responses in all models studied (31). Studies have also demonstrated that IFNAR signaling can to date, including donor RBCs expressing hen egg lysozyme (HEL), promote antiviral neutralizing Ab responses (32, 33). In addition, the human KEL2 Ag (K2), and a chimeric protein containing IFN-a/b has been implicated in the pathogenesis of multiple auto- HEL, OVA, and the Duffy Ag (HOD) (20, 24, 25). immune diseases, including rheumatoid arthritis, dermatomyositis, Poly(I:C) promotes innate and adaptive immune responses scleroderma, and Sjo¨gren syndrome (34–38). In particular, many through multiple pathways. Poly(I:C) is recognized by multiple patients with SLE have elevated serum IFN-a/b and IFN-stimulated pattern recognition receptors, including TLR3, and the retinoic acid gene expression, which correlate with increased autoantibody inducible gene-I (RIG-I)–like receptors (RLRs), which include production and disease severity (39–42). More than 50% of SLE- melanoma differentiation–associated gene 5 (MDA5) and RIG-I associated genetic variants have been linked to the IFN-a/b pathway (26–28). Signaling through TLR3 and RLRs use distinct signaling (43), and clinical trials of anti–IFN-a therapy are in progress (44, 45). adapter proteins to induce type I IFNs (IFN-a/b) and numerous Given that multiple inflammatory conditions are associated with NFҡB-regulated , including IL-6, IL-12, macrophage elevated IFN-a/b production and RBC alloimmunization, we hy- chemoattractant protein, and TNF-a (29, 30). Thus, poly(I:C) may pothesize that IFN-a/b may regulate inflammation-induced alloim- Downloaded from augment RBC alloimmune responses by activating multiple mune responses to transfused RBC Ags. The antithetical K1 and K2 pathways that induce critical inflammatory cytokines. However, Ags in the Kell system are defined by a methionine or threonine at the relative roles of each pathway and cytokine in inflammation- position 193, respectively. K1 is the most immunogenic RBC Ag that induced RBC alloimmunization have not been investigated. is not routinely matched between donor units and recipients (46, 47). Of the many inflammatory cytokines that may regulate With the exception of phenotypic matching or antigenic avoidance,

inflammation-induced alloimmunization, IFN-a/b stands out as an there are no therapies that prevent K1 alloimmunization in humans http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 1. KEL transgenic mice ex- press the human KEL glycoprotein spe- cifically on RBCs. (A–C) Flow cytometric analysis of K1 expression on peripheral blood (A) Ter119+ RBCs and (B)CD41+ platelets, and (C) spleen CD45+ leukocytes and RBCs of K1 and WT mice. Numbers on representative dot plots (left) from K1 mice indicate the percent of cells within the drawn gate. (B) Platelet-rich plasma was gated on CD452 Ter1192 cells. (A–C) Histograms are gated on dot plot gates. Representative of three independent experiments. The Journal of Immunology 1043

(48). In this study, we introduce a new transgenic donor mouse that ex- cotransfused with K1 RBCs and recombinant mouse IFN-a (HC1040; 3 pressesthehumanK1AgonRBCsandformallytesttheroleofIFN-a/b Hycult Biotech, the Netherlands) at doses of 2–100 3 10 units in PBS. A 3 3 production and IFNAR signaling in a model of inflammation- total of 20 10 units of recombinant IFN-a (rIFN-a) approximates poly (I:C)-induced measured serum IFN-a (∼10 3 103 units per ml in 2 ml blood induced RBC alloimmunization. volume) at the time of infusion. Materials and Methods Detection of alloantibodies Mice Abs produced against the transgenic Kell glycoprotein (K1 variant) are described as anti-K1 IgG and were measured by flow-cytometric crossmatch C57BL/6 and congenic C57BL/6-Ly5.1 wild-type (WT) mice were pur- mob/mob 7, 14, 21, and 28 d after transfusion as previously described (24). K1 or WT chased from Charles River Laboratories (Wilmington, MA). IFNb RBCs were incubated with serum from transfused mice and subsequently mice were purchased from the Jackson Laboratory (Bar Harbor, ME). 2/2 2/2 2/2 2/2 2/2 2/2 stained for RBC-bound IgG (goat anti-mouse IgG APC; Jackson Ifnar1 , Mavs , Irf3 , Irf7 , MyD88 , and Trif mice were ImmunoResearch, West Grove, PA). The adjusted MFI was calculated by previously described (49–54). Appropriate gene-deficient mice were bred 2/2 2/2 2/2 subtracting the reactivity of serum with syngeneic WT RBCs from the to produce Irf3/7 and MyD88 Trif double knockout mice. All reactivity of serum with K1 RBCs. To maximize detection sensitivity, mice were 8–12 wk of age and were backcrossed to the C57BL/6 back- serum was not diluted. Figure data illustrates the peak Ab response, 28 d ground for more than eight generations. All animal protocols were ap- following transfusion. Flow cytometry of RBCs was performed using a BD proved by the Yale Institutional Animal Care and Use Committee. FACSCalibur (San Jose, CA) and analyzed using FlowJo software (Tree Transgenic mice expressing the human Kell glycoprotein consisting of Star, Ashland, OR). the K1 variant (K1 mice) were generated as previously described (55). Briefly, K1 mice were produced by amplifying K2 cDNA from a human Flow cytometry bone marrow cDNA library. K2 was mutated to K1 using a QuickChange mutagenesis (Stratagene, Santa Clara, CA) and inserted into a previ- For detection of K1 expression, splenocytes, peripheral blood, and platelet- Downloaded from ously described RBC expression vector, containing the murine b-globin rich plasma were stained with a monoclonal anti-Kell Ab (Mima-8) (58) promoter, the b-globin locus control region, and the b-globin intron 2 and followed by anti-mouse IgG and Abs against cell type–specific markers. b 39 enhancers (56, 57). Following sequencing to rule out the introduction of Mima-8 recognizes the Js epitope on the human Kell glycoprotein, which other mutations, the construct was injected into fertilized embryos, which is expressed by the K1 transgene. A combination of Mima-8 and Mima-9, b were implanted into pseudopregnant C57BL/6 mice. The same approach which recognizes Kp epitopes of the K1 transgene, was used to compare was described to generate K2 and K1 transgenic mice previously. As the K1 expression by KEL1A and K1 mice. Platelet-rich plasma was gen- erated by centrifuging peripheral blood at 8000 3 g for 10 min. For

initially described K1 transgenic mouse, previously published as KEL1A, http://www.jimmunol.org/ had a low expression of the transgene, additional resources were invested analysis of dendritic cells (DCs), spleens were minced with a razor blade to generate new transgenic founders, including the K1 mouse used in this and filtered through 100 mm Nylon mesh prior to RBC lysis. Single-cell study (55). suspensions were stained with fluorescently conjugated Abs specific for cell-surface proteins, including CD19 (Clone: 6D5), TCRb (H57- Inflammation-induced alloimmunization 597), and I-A/I-E [MHC class II (MHCII), M5/114.15.2], CD86 (GL-1), Ly6C (HK1.4) and F4/80 (BM8) from BioLegend (San Diego, CA); Recipient mice were injected i.p. with 100 mg poly(I:C) (InvivoGen, San CD45.1 (A20), CD11c (N418), CD11b (M1/70), CD8a (53-6.7), Ter- Diego, CA) at indicated time points relative to transfusion on day 0. Pe- 119, and Siglec H (eBio440c) from eBioscience (San Diego, CA), and ripheral blood of K1 mice was collected in 12% citrate phosphate dextrose CD41 (MWReg30) from BD Biosciences (San Jose, CA). Zombie-NIR adenine (Jorgensen Labs, Melville, NY), leuko-reduced with a Pall syringe (BioLegend) was used to exclude dead cells. Cells were acquired with a filter (East Hills, NY), and washed with PBS. Recipient WT, chimeric, or Miltenyi MACSQuant flow cytometer and analyzed using FlowJo. by guest on September 23, 2021 gene-deficient mice were transfused in the lateral tail vein with 75 mlof packed RBCs, the approximate mouse equivalent of 1 U of human RBCs. Generation of bone marrow chimeras For CD4+ T cell depletion, 4 and 2 d prior to transfusion, recipient mice were injected i.p. with 200 mg GK1.5 Ab (Bio X Cell, West Lebanon, NH) Recipient WT C57BL/6 (CD45.2+) and Ifnar12/2 (CD45.2+) mice were or an isotype matched control. For in vivo IFN-a treatment, WT mice were exposed twice to x-ray irradiation (6.35 Gy, 3 h apart) using an X-RAD

FIGURE 2. Poly(I:C) induces anti-K1 alloim- mune responses. WT mice were injected with or withoutpoly(I:C)andtransfusedwithK1RBCs. Serum anti-K1 IgG 28 d following transfusion was measured by flow-cytometric cross-match. (A) Representative histograms of flow cytometric cross-match. WT and K1 RBCs were incubated with serum of transfusion recipients, followed by anti-mouse IgG. (B) Quantified anti-K1 IgG. The adjusted MFI was calculated by subtract- ingtheMFIofWTRBCsfromtheMFIofK1 RBCs. (A and B) Poly(I:C)-treated mice were injected i.p. 3 h prior to transfusion. (C)Poly(I:C) was administered at varying time points relative to K1 RBC transfusion on day 0. A dash (-) indicates no poly(I:C) treatment. Representative of three independent experiments with four to five mice per group. Bars indicate the mean of anti-K1 IgG levels from individual mice, indicated by circles. *p , 0.05. n.s., not significant by Kruskal–Wallis test with Dunn posttest. 1044 IFN-a/b REGULATES RBC ALLOIMMUNIZATION

320 irradiator (Precision X-ray, North Branford, CT). Recipients were The anti-K1 IgG response was measured by flow cytometric cross- injected i.v. with 3 3 106 bone marrow cells from WT C57BL/6-Ly5.1 + 2/2 match. Although no Ab response to K1 was detected in the absence of (CD45.1 )orIfnar1 mice 2–4 h after irradiation. Peripheral blood was poly(I:C), recipients treated with poly(I:C) 3 h prior to transfusion analyzed for lymphocyte reconstitution 6 wk after bone marrow transfer. CD45.1 and CD45.2 congenic markers were used to identify the source of produced anti-K1 IgG alloantibodies (Fig. 2A, 2B, Supplemental Fig. reconstituted cells. Mice were transfused 8–9 wk following bone marrow 2). To begin examination of mechanisms underlying poly(I:C)- reconstitution. induced alloimmunization, we assessed the alloimmune responses Measurement of IFN-a/b of WT mice treated with poly(I:C) at varying time points before or after transfusion on day 0. As shown in Fig. 2C, poly(I:C) Serum IFN-a was measured by ELISA as previously described (59). For treatment 3 h prior to transfusion induced maximal alloimmunization. mRNA measurement, splenocytes were enriched for DCs using a mouse In contrast, treatment one or more days before or after transfusion did pan-DC enrichment kit (19763; Stemcell Technologies, Vancouver, BC). Enrichment was examined by flow cytometry. mRNA was isolated with a not induce significant anti-K1 alloantibody production, compared Qiagen RNEasy MiniKit (Valencia, CA), treated with DNAse, and reverse- with untreated controls. Thus, poly(I:C) administered in the imme- transcribed with a Roche Applied Sciences kit (Indianapolis, IN). cDNA diate peri-transfusion period induces anti-K1 alloimmunization. was quantitated with a KAPA SYBR FAST qPCR kit (KAPA Biosystems, Wilmington, MA), using a Stratagene Mx3000P instrument. Primers for IFNAR signaling in hematopoietic cells is required for Ifna4 and Ifnb are: Ifna4 forward, 59-CTG CTA CTT GGA ATG CAA inflammation-induced RBC alloimmunization CTC-39; Ifna4 reverse, 59-CAG TCT TGC CAG CAA GTT GG-39; Ifnb forward, 59-GCA CTG GGT GGA ATG AGA CTA TTG-39; Ifnb reverse, One of the earliest phases of humoral immune responses is the 59-TTC TGA GGC ATC AAC TGA CAG GTC-39. production of innate cytokines that promote activation of APCs,

Western blot analysis including DCs. Although poly(I:C) induces multiple inflammatory Downloaded from cytokines, IFN-a/b has been shown to regulate DC activation and Peripheral blood cells from K1 and C57BL/6 WT mice were lysed using RBC consumption in other models (60–62). Thus, immediately hypotonic sodium phosphate. Samples were reduced, electrophoresed on a prior to transfusion, we measured serum IFN-a of mice pretreated polyacrylamide gel, and blotted to nitrocellulose membranes. The KEL glycoprotein was detected using the mouse mAb, MM0435-12 3 3 (Novus with poly(I:C) at varying time points. Compared to untreated Biologicals, Littleton, CO) followed by goat anti-mouse IgG1 HRP mice, serum IFN-a was only significantly increased in mice treated (Bethyl Laboratories, Montgomery, TX). Detection of b-actin was used as

with poly(I:C) 3 h prior to transfusion (Fig. 3A). http://www.jimmunol.org/ a loading control. Bands were detected with Immobilon Western Chemi- To determine whether IFN-a/b plays a role in inflammation- luminescent HRP Substrate (Millipore, Darmstadt, Germany). induced alloimmunization, we examined RBC alloimmune responses Statistics Statistical analyses were performed using Graph Pad Prism software (San Diego, CA). Statistical significance between two groups was determined using a Mann–Whitney U test or an unpaired two-sided Student t test for nonparametric and parametric data, respectively. Significance between three or more groups was determined using a Kruskal–Wallis test with a Dunn posttest. Alloantibody data were analyzed with nonparametric tests. For all bar graphs, bars indicate the mean of data from individual mice, by guest on September 23, 2021 which are indicated by circles. Results KEL transgenic mice express the human KEL glycoprotein specifically on RBCs To examine inflammation-induced alloantibody responses to the K1 Ag from the Kell system, we generated donor transgenic mice that express the Kell glycoprotein (K1 variant) on RBCs (K1 mice). K1 cDNA was inserted into a vector containing the murine b-globin promoter and enhancer regions to promote erythroid-specific transgene expression (56, 57). Multiple founder lines were generated, including K1 mice and the previously described KEL1A mice (55). Using monoclonal Abs against the Jsb epitopes (MIMA-8) and Kpb epitopes (MIMA-9) expressed by the K1 transgene, we observed higher expression on K1 RBCs, compared with KEL1A RBCs (Supplemental Fig. 1A). Given this result, we used K1 mice for subsequent experiments. Western blot analysis demonstrated expression of K1 on peripheral blood cells of K1 mice (Supplemental Fig. 1B). We then examined K1 cell surface expression on RBCs, platelets, and leukocytes. As showninFig.1A,K1isexpressedbyTer119+ RBCs in the peripheral blood of K1 mice. However, K1 mice do not express K1 on CD41+ platelets in peripheral blood or CD45+ leukocytes in the spleen (Fig. FIGURE 3. IFNAR signaling in hematopoietic cells is required for in- A C 1B, 1C). Thus, hematopoietic cells of K1 mice express the human flammation-induced K1 RBC alloimmunization. ( and ) IFN-a in serum of mice treated with poly(I:C) at the (A) indicated times or (C) 3 h prior to KEL glycoprotein in an erythroid-specific manner. analysis by ELISA. (B and D) Anti-K1 IgG in serum of indicated mice 28 d Inflammation induces alloimmune responses to transfused following transfusion with K1 RBCs. Mice were administered poly(I:C) 3 h K1-expressing RBCs prior to transfusion. (D) Bone marrow chimeras were generated by transferring donor bone marrow into irradiated recipient mice 8–9 wk prior to transfusion. To assess the alloimmune response to K1-expressing RBCs (K1 Representative of two (A, C,andD)andthree(B) independent experiments RBCs), RBCs from K1 mice were leuko-reduced and transfused with four to five mice per group. *p , 0.05. n.s., not significant by Mann– into WT controls in the presence or absence of poly(I:C) treatment. Whitney U test, **p , 0.01 by Kruskal–Wallis test with Dunn posttest. The Journal of Immunology 1045 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 4. IFN-a/b regulates poly(I:C)-induced activation of cDCs. Indicated mice were transfused with K1 RBCs. (A and C) Flow cytometric analysis of spleen CD11chi MHCII+ cDCs from (A)WTor(C) indicated mice transfused 5 h prior to analysis and injected i.p. with or without poly(I:C) 3 h prior to transfusion. Cells are gated on live CD192 TCRb2 splenocytes. Numbers on dot plots indicate the percent of cells within the drawn gate. (B) CD86 expression of spleen cDCs of WT mice untreated or treated with poly(I:C) at the indicated time point. (D and E) CD86 expression of spleen cDCs gated in (C). (F) Serum anti-K1 IgG of isotype control (iso) and GK1.5 (anti-CD4) Ab treated WT mice 28 d following transfusion and poly(I:C) injection. Representative of three (A, B, and F) or two (C–E) independent experiments with three to five mice per group. *p , 0.05 by Mann–Whitney U test. (E) ***p , 0.001 by unpaired two-sided Student t test. in mice lacking the only known receptor for IFN-a/b (Ifnar12/2). WT bone marrow rescued the anti-K1 IgG response. However, the Following poly(I:C) treatment and transfusion, production of anti-K1 alloimmune response of WT mice reconstituted with Ifnar12/2 2 2 alloantibodies by Ifnar1 / mice was significantly diminished, com- bone marrow was completely abrogated. Collectively, these results 2 2 pared to WT controls (Fig. 3B). Notably, WT and Ifnar1 / mice demonstrate that IFNAR signaling in hematopoietic cells is required contained comparablelevelsofserumIFN-a at the time of transfu- for inflammation-induced K1 RBC alloimmunization. sion (Fig. 3C). Thus, the decreased alloimmune response in Ifnar12/2 IFN-a/b promotes DC activation during T cell–dependent mice is not due to differences in poly(I:C)-induced IFN-a production. anti-K1 alloimmune responses However, the diminished response could be due to altered lymphoid structure in Ifnar12/2 mice. To address this possibility, we assessed Recent studies in other transfusion models have demonstrated that alloimmune responses in bone marrow chimeric mice generated by T cell–dependent alloimmune responses to RBC Ags require Ag reconstituting irradiated recipient mice with WT or Ifnar12/2 bone presentation by activated conventional DCs (cDCs) (17, 63). To marrow. As shown in Fig. 3D, reconstitution of Ifnar12/2 mice with determine whether poly(I:C)-mediated inflammation promotes 1046 IFN-a/b REGULATES RBC ALLOIMMUNIZATION Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 5. Poly(I:C)-induced IFN-a/b is produced by CD8+ cDCs. IFN-bmob/mob mice were injected with poly(I:C) or PBS 8 h prior to analysis. (A) Representative flow cytometric analysis of IFN-b/YFP expression by spleen CD11chi cDCs, MHCII+ cells, and CD11b+ or Ly6C+ monocytes. Gated on live CD192 TCRb2 nonlymphocytes. (B) Representative flow cytometric analysis of spleen CD8a+ and CD11b+ cDCs (right) from (Figure legend continues) The Journal of Immunology 1047 cDC activation during alloimmunization, we measured expression also represented by an increase in total IFN-b/YFP expression by of the activation marker, CD86, by spleen CD11chi MHCII+ cDCs CD8a+ cDCs (Supplemental Fig. 4C, 4D). These results indicate that from WT mice pretreated with poly(I:C) at varying time points. a subset of CD8a+ cDCs produce IFN-a/b during inflammation- Six hours following transfusion with K1 RBCs, cDCs from mice induced K1 alloimmunization. untreated or treated with poly(I:C) 1 or 7 d prior to transfusion expressed comparable levels of CD86. In contrast, cDCs from Mitochondrial antiviral signaling protein–dependent IFN-a/b mice treated with poly(I:C) 3 h prior to transfusion had elevated production is required for K1 RBC alloimmunization CD86 expression (Fig. 4A, 4B). To determine whether the increase The abrogated anti-K1 response in Ifnar12/2 mice indicates that in CD86 expression was mediated by IFNAR signaling, CD86 ex- IFN-a/b production may also be required for alloimmunization. 2/2 pression was also assessed in Ifnar1 mice treated with or without As shown in Fig. 6A, poly(I:C) can induce IFN-a/b by binding poly(I:C) 3 h prior to transfusion. Compared to WT mice, CD86 endosomal TLR3, which utilizes the adaptor protein, TIR-domain– 2/2 upregulation by cDCs from poly(I:C)-treated Ifnar1 mice containing adaptor protein inducing IFN-b (TRIF), to mediate was significantly reduced (Fig. 4C–E). Hence, IFNAR signaling downstream signaling (28). MDA5 and RIG-I in the cytosol can regulates cDC activation during inflammation-induced K1 also recognize poly(I:C) and use the signaling protein, mitochondrial alloimmunization. antiviral signaling protein (MAVS), to induce IFN-a/b (27). Both Given that cDC activation enhances Ag presentation to T cells, pathways converge upon the canonical transcription factors in the we determined whether production of anti-K1 alloantibodies re- nucleus, IFN regulatory factor (IRF) 3 and IRF7 (31). quires T cell help. Treatment of mice with the anti-CD4 Ab, GK1.5, To determine which pathway induces IFN-a/b production

+ Downloaded from has been shown to deplete CD4 T cells, which remained unde- during alloimmunization, we measured serum IFN-a 3 h follow- tectable for at least 21 d (64). WT mice were pretreated with ing poly(I:C) treatment in mice lacking MAVS (Mavs2/2), IRF3 GK1.5 or an isotype control Ab prior to poly(I:C) treatment 3 h and IRF7 (Irf3/72/2), or the entire TLR pathway (MyD882/2 before transfusion. Compared to control mice, the alloimmune Trif2/2). WT and MyD882/2 Trif2/2 mice produced comparable response of GK1.5-treated mice was fully abrogated (Fig. 4F). levels of IFN-a. However, levels of IFN-a produced by Mavs2/2 Collectively, these results indicate that IFN-a/b may regulate K1 and Irf3/72/2 mice were significantly diminished, compared with

T cell–dependent alloimmune responses, at least in part, by pro- WT mice (Fig. 6B). To determine whether IFN-a/b production http://www.jimmunol.org/ moting cDC activation. regulates inflammation-induced alloimmunization, we assessed Poly(I:C)-mediated inflammation induces IFN-a/b production anti-K1 alloimmune responses in these knockout mice. Although 2/2 2/2 by CD8a+ cDCs WT and MyD88 Trif mice produced similar levels of anti- K1 IgG, alloimmune responses by Mavs2/2 and Irf3/72/2 were cDCs and plasmacytoid DCs (pDCs) have been shown to produce significantly diminished (Fig. 6C). Collectively, these results indicate IFN-a/b in response to inflammatory stimuli (28, 65). Hence, to that IFN-a/b production is required for K1 RBC alloimmunization. determine whether DCs produce IFN-a/b during inflammation- Additionally, although TLR3-mediated signaling is not required for induced alloimmune responses, we measured IFN-a/b mRNA in poly(I:C)-induced IFN-a/b production or alloimmunization, MAVS- WT splenocytes enriched for DCs by magnetic cell selection dependent signaling is required. by guest on September 23, 2021 (Supplemental Fig. 3A). In contrast to PBS treated mice, DC-enriched splenocytes from poly(I:C)-treated mice produced IFN-a and IFN-b IFN-a is sufficient to induce alloimmunization to transfused mRNA 8 h following treatment (Supplemental Fig. 3B, 3C). K1 RBCs + + DCs are comprised of multiple CD11c MHCII DC-subset Although IFN-a/b production and signaling are required for anti- + + + populations, including CD11b and CD8a cDCs and Siglec H K1 alloimmune responses, poly(I:C) may promote alloimmuni- pDCs. To determine which DCs produce IFN-a/b during poly(I:C)- zation by inducing multiple cytokines. Thus, to determine whether induced alloimmunization, we used IFN-b reporter mice that IFN-a/b is sufficient to induce alloimmunization in the absence of produce the yellow fluorescent protein (YFP) transcript linked to poly(I:C), we assessed alloimmune responses of WT mice mob/mob endogenous IFN-b mRNA (IFN-b mice) (65). Scheu et al. cotransfused with K1 RBCs and rIFN-a. As shown in Fig. 6D, (65) reported that YFP expression was not observed in poly(I:C)- rIFN-a treatment induced anti-K1 alloantibody responses in a mob/mob treated IFN-b mice until 6 h after treatment. Given that DC dose-dependent manner. Thus, IFN-a is sufficient to induce K1 activation typically peaks 6–8 h following treatment with inflam- RBC alloimmunization. matory stimuli (66), IFN-b/YFP expression was analyzed 8 h fol- lowing treatment. Poly(I:C) treatment of IFN-bmob/mob mice induced IFN-b/YFP expression in a low percentage of CD11c+ and Discussion MHCII+ cells, but not CD11b+ Ly6C+ monocytes (Fig. 5A) Multiple studies have established that certain inflammatory dis- or lymphocytes (Supplemental Fig. 4A, 4B). Analysis of CD11c+ orders and inflammatory stimuli increase the frequency and MHCII+ DC subsets demonstrated that spleen CD11b+ cDCs and magnitude of alloimmune responses to RBC Ags (12–16, 19–21). Siglec H+ pDCs from poly(I:C) and PBS-injected mice expressed However, the molecular mechanisms underlying these findings comparable levels of IFN-b/YFP (Fig. 5B–E, Supplemental Fig. have not been previously studied. We demonstrate that IFN-a/b 4C, 4D). However, poly(I:C) treatment resulted in a significant in- production and IFNAR signaling are required for alloimmune crease in the percentage of IFN-b/YFP-expressing CD8a+ cDCs, responses to the K1 Ag in a murine model of inflammation-induced compared with PBS-injected mice (Fig. 5D, 5E). This result was alloimmunization. Although poly(I:C)-induced alloimmunization

poly(I:C)-treated IFN-bmob/mob mice, gated on total splenic cDCs (left). (C) Representative analysis of spleen CD11c+ Siglec H+ pDCs, gated on live CD192 TCRb2 cells. (D) IFN-b/YFP+ cDCs, and pDCs, gated as in (B) and (C). (E) Summary data of IFN-b/YFP+ cells identified in (D). (A–D) Numbers on plots indicate percent of cells within the drawn gate. Representative of three independent experiments with four to five mice per group. *p , 0.05. n.s., not significant by unpaired two-sided Student t test. 1048 IFN-a/b REGULATES RBC ALLOIMMUNIZATION

was initially described years ago (20), the receptor-associated pathways that recognize poly(I:C) and induce RBC alloimmune responses have not been understood until now. In this manuscript, we show that MAVS-mediated pathways are required for poly(I:C)- induced IFN-a/b production and alloimmunization. Further, we report that IFN-a, in the absence of an adjuvant, is sufficient to induce RBC alloimmunization. Assessing the alloimmune response of transfusion recipients exposed to inflammatory stimuli at varying times provides insight into the mechanisms underlying inflammation-induced alloimmunization. Previous studies in other murine transfusion models have shown that immediate pretreatment or cotrans- fusion of pathogen-associated molecular patterns can enhance RBC alloimmunization (20, 21). The results of the current study further indicate that the recipient’s inflammatory state at the time of transfusion can dictate the immune response to RBC alloantigens. Treatment of recipient mice with poly(I:C), only during the peri-transfusion period, induced cDC activa- Downloaded from tion and T cell–dependent alloimmunization to the human K1 Ag. These findings agree with prior studies demonstrating a critical role for cDC activation in T cell–dependent alloim- mune responses to stored RBCs expressing the HOD Ag (18, 63). A recent study reported that the timing of poly(I:C) treatment influences alloantibody responses to transfused HOD RBCs (19). However, in contrast to our findings, the anti-HEL http://www.jimmunol.org/ alloimmune response of mice treated with poly(I:C) 7 d prior to HOD RBC transfusion was significantly higher than those treated just 4 h prior to transfusion. These somewhat conflicting findings may be due to qualitative or quantitative differences in the response to different RBC alloantigens. Whereas transient MAVS-mediated IFN-a/b production is critical to induce K1 alloimmunization in part by activating DCs, anti-HEL alloantibody responses may be regu-

lated by alternate mechanisms. by guest on September 23, 2021 In contrast to alloimmune responses to HOD or K2 RBCs (25, 24), we found that anti-K1 responses only occurred following treatment with inflammatory stimuli. Thus, the K1 RBC model allowed for direct evaluation of inflammatory pathways critical for alloimmunization. Examination of alloimmune responses in Ifnar12/2 mice and bone marrow chimeras revealed that IFNAR signaling in hematopoietic cells was required for K1 alloim- munization. Given that Ifnar12/2 and WT mice were previously reported to produce comparable IgM and IgG responses to im- munization with multiple soluble T dependent Ags (49), this result was unlikely due to altered hematopoiesis in Ifnar12/2 mice. Rather, it is likely that IFNAR signaling in multiple hema- topoietic cell types promotes K1 alloimmunization. We show that IFNAR signaling regulates inflammation-induced cDC activation following transfusion. Additionally, IFN-a/b has been shown to di- rectly promote activation of other cell types, including lymphocytes, during T-dependent humoral immune responses (31). Results of experiments in Ifnar12/2 mice indicated that IFN-a/b production may also be necessary for K1 alloimmunization. Indeed, the only WT mice that produced anti-K1 alloantibodies were transfused in the presence of elevated serum IFN-a.Moreover,

mice were injected with poly(I:C) 3 h prior to (B) analysis or (C) transfusion with K1 RBCs. (B) IFN-a in serum of indicated mice. (C and D) Anti-K1 IgG in serum of indicated mice 28 d following transfusion. (D) WT mice FIGURE 6. IFN-a/b production is necessary and sufficient for K1 RBC were cotransfused with K1 RBCs and rIFN-a. Representative of two in- alloimmunization. (A) Schematic of pathways leading to IFN-a/b pro- dependent experiments with four to five mice per group. (B–D)*p , 0.05 duction. Recognition of poly(I:C) by pattern recognition receptors acti- by Mann–Whitney U test. MyD88, myeloid differentiation primary re- vates multiple pathways leading to IFN-a/b induction. (B and C) Indicated sponse gene 88. The Journal of Immunology 1049 following poly(I:C) treatment, mice lacking the canonical IFN-a/b 4. Hoeltge, G. A., R. E. Domen, L. A. Rybicki, and P. A. Schaffer. 1995. Multiple red a cell transfusions and alloimmunization. Experience with 6996 antibodies detected in transcription factors, IRF3 and IRF7, were unable to produce IFN- a total of 159,262 patients from 1985 to 1993. Arch. Pathol. Lab. Med. 119: 42–45. or anti-K1 alloantibodies. In agreement with prior studies in other 5. Redman, M., F. Regan, and M. Contreras. 1996. A prospective study of the inci- models (60, 65), we observed that a fraction of CD8a+ cDCs are dence of red cell allo-immunisation following transfusion. Vox Sang. 71: 216–220. 6. Vichinsky, E. P., A. Earles, R. A. Johnson, M. S. Hoag, A. Williams, and the primary hematopoietic source of poly(I:C)-induced IFN-b.Al- B. Lubin. 1990. Alloimmunization in sickle cell anemia and transfusion of ra- though TLR3 has been shown to mediate poly(I:C)-induced IFN-a/b cially unmatched blood. N. Engl. J. Med. 322: 1617–1621. production (28), TLR3 signaling was dispensable for IFN-a pro- 7. Yazdanbakhsh, K., R. E. Ware, and F. Noizat-Pirenne. 2012. Red blood cell alloimmunization in sickle cell disease: pathophysiology, risk factors, and duction and K1 alloimmunization. Given that TLR3 is preferentially transfusion management. Blood 120: 528–537. expressed in murine spleen CD8a+ cDCs (67), this result was un- 8. (FDA), U. D. O. H. A. H. S. 2014. Fatalities reported to the FDA following blood likely due to lack of TLR3 expression in IFN-a/b–producing cells. collection and transfusion: annual summary for fiscal year 2014. Available at: http://www.fda.gov/downloads/biologicsbloodvaccines/safetyavailability/ The finding that poly(I:C)-induced IFN-a/b production is MAVS- reportaproblem/transfusiondonationfatalities/ucm459461.pdf. Accessed: November dependent indicates that poly(I:C) gainsaccesstothecytosolin 11, 2015. CD8a+ cDCs and stimulates RLRs. Thus, we conclude that cytosolic 9. Ko¨rmo¨czi, G. F., and W. R. Mayr. 2014. Responder individuality in red blood + cell alloimmunization. Transfus. Med. Hemother. 41: 446–451. RLRs of spleen CD8a cDCs recognize poly(I:C) and induce MAVS- 10. Nickel, R. S., J. E. Hendrickson, R. M. Fasano, E. K. Meyer, A. M. Winkler, dependent IFN-a/b production that is required for alloimmunization. M. M. Yee, P. A. Lane, Y. A. Jones, F. D. Pashankar, T. New, et al. 2016. Impact of red blood cell alloimmunization on sickle cell disease mortality: a case series. Although not addressed in this study, the dependence of K1 Transfusion 56: 107–114. alloimmunization on IFN-a/b production and IFNAR signaling 11. Telen, M. J., A. Afenyi-Annan, M. E. Garrett, M. R. Combs, E. P. Orringer, and does not rule out a contributory role for NFҡB cytokines induced A. E. Ashley-Koch. 2015. Alloimmunization in sickle cell disease: changing antibody specificities and association with chronic pain and decreased survival. by poly(I:C). It is plausible that a lack of IFNAR signaling in Transfusion Downloaded from 2 2 55: 1378–1387. Ifnar1 / mice may inhibit production of other critical cyto- 12. Fasano, R. M., G. S. Booth, M. Miles, L. Du, T. Koyama, E. R. Meier, and kines. However, Salem et al. (29) reported that poly(I:C) treat- N. L. Luban. 2015. Red blood cell alloimmunization is influenced by recipient 2/2 inflammatory state at time of transfusion in patients with sickle cell disease. ment of WT and Ifnar1 mice induced comparable levels of Br. J. Haematol. 168: 291–300. TNF-a, macrophage chemoattractant protein, IL-6, and IFN-g. 13. Papay, P., K. Hackner, H. Vogelsang, G. Novacek, C. Primas, W. Reinisch, Treatment with rIFN-a was sufficient to induce K1 alloimmu- A. Eser, A. Mikulits, W. R. Mayr, and G. F. Kormoczi. 2012. High risk of transfusion-induced alloimmunization of patients with inflammatory bowel nization, yet other cytokines may augment the quality or magnitude disease. Am J Med. 125: 717.e1–8. http://www.jimmunol.org/ of the response. IL-6 was recently shown to enhance alloimmuni- 14. Yazer, M. H., D. J. Triulzi, B. Shaz, T. Kraus, and J. C. Zimring. 2009. Does a febrile reaction to platelets predispose recipients to red blood cell alloimmuni- zation to stored HOD RBCs by promoting T follicular helper cell zation? Transfusion 49: 1070–1075. differentiation (17). The role of other inflammation-induced cyto- 15. Ramsey, G., and S. J. Smietana. 1995. Multiple or uncommon red cell alloanti- kines in alloimmunization will be the subject of future study. bodies in women: association with autoimmune disease. Transfusion 35: 582–586. 16. Evers, D., J. G. van der Bom, J. Tijmensen, R. A. Middelburg, M. de Haas, Clinically significant alloantibodies are formed in only 3–10% of S. Zalpuri, K. M. de Vooght, D. van de Kerkhof, O. Visser, N. C. Pe´que´riaux, transfusion recipients, and the majority of transfusion recipients et al. 2016. Red cell alloimmunisation in patients with different types of in- never form detectable RBC alloantibodies (22). Given the role of fections. Br. J. Haematol. 175: 956–966. 17. Arneja, A., J. E. Salazar, W. Jiang, J. E. Hendrickson, J. C. Zimring, and inflammation in promoting alloimmunization, these nonresponders C. J. Luckey. 2016. -6 receptor-alpha signaling drives anti-RBC al- may have been initially transfused in the absence of inflammatory loantibody production and T-follicular helper cell differentiation in a murine by guest on September 23, 2021 stimuli. Consistent with this notion, prior studies in some murine model of red blood cell alloimmunization. Haematologica 101: e440–e444. 18. Gibb, D. R., S. Calabro, D. Liu, C. A. Tormey, S. L. Spitalnik, J. C. Zimring, models have shown that transfusion of allogeneic RBCs in the absence J. E. Hendrickson, E. A. Hod, and S. C. Eisenbarth. 2016. The Nlrp3 inflamma- of an inflammatory stimulus induces long-term nonresponsiveness to some does not regulate alloimmunization to transfused red blood cells in mice. EBioMedicine 9: 77–86. the Ag (68). Although not directly tested, transfusion of K1 RBCs in 19. Elayeb, R., M. Tamagne, P. Bierling, F. Noizat-Pirenne, and B. Vingert. 2016. the absence of inflammation may have a similar result. In this study, Red blood cell alloimmunization is influenced by the delay between Toll-like demonstration that cotransfusion of rIFN-a is sufficient to prevent receptor agonist injection and transfusion. Haematologica 101: 209–218. 20. Hendrickson, J. E., M. Desmarets, S. S. Deshpande, T. E. Chadwick, nonresponsiveness indicates that exposure to IFN-a/b–inducing C. D. Hillyer, J. D. Roback, and J. C. Zimring. 2006. Recipient inflammation stimuli during transfusion may be one factor that dictates responder affects the frequency and magnitude of immunization to transfused red blood versus nonresponder status. cells. Transfusion 46: 1526–1536. 21. Yu, J., S. Heck, and K. Yazdanbakhsh. 2007. Prevention of red cell alloimmuni- In summary, we report that MAVS-mediated IFN-a/b produc- zation by CD25 regulatory T cells in mouse models. Am. J. Hematol. 82: 691–696. tion and IFNAR signaling are required for alloimmune responses 22. Ryder, A. B., J. C. Zimring, and J. E. Hendrickson. 2014. Factors influencing to the human K1 Ag in a murine model of inflammation-induced RBC alloimmunization: lessons learned from murine models. Transfus. Med. Hemother. 41: 406–419. alloimmunization. These findings provide a potential mechanistic 23. Bao, W., J. Yu, S. Heck, and K. Yazdanbakhsh. 2009. Regulatory T-cell status in basis for past observations of inflammation-induced alloimmuni- red cell alloimmunized responder and nonresponder mice. Blood 113: 5624–5627. 24. Stowell, S. R., K. R. Girard-Pierce, N. H. Smith, K. L. Henry, C. M. Arthur, zation. If they extend to human studies, patients with IFN-a/b– J. C. Zimring, and J. E. Hendrickson. 2014. Transfusion of murine red blood associated conditions and patients receiving IFN-a/b therapy may cells expressing the human KEL glycoprotein induces clinically significant al- have an elevated risk of alloimmunization and may benefit from loantibodies. Transfusion 54: 179–189. 25. Hendrickson, J. E., E. A. Hod, S. L. Spitalnik, C. D. Hillyer, and J. C. Zimring. personalized transfusion protocols, including extended Ag matching. 2010. Storage of murine red blood cells enhances alloantibody responses to an erythroid-specific model antigen. Transfusion 50: 642–648. 26. Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu, T. Imaizumi, Disclosures M. Miyagishi, K. Taira, S. Akira, and T. Fujita. 2004. The RNA helicase RIG-I The authors have no financial conflicts of interest. has an essential function in double-stranded RNA-induced innate antiviral re- sponses. Nat. Immunol. 5: 730–737. 27. Gitlin, L., W. Barchet, S. Gilfillan, M. Cella, B. Beutler, R. A. Flavell, References M. S. Diamond, and M. Colonna. 2006. Essential role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis 1. Blumberg, N., K. Peck, K. Ross, and E. Avila. 1983. Immune response to chronic picornavirus. Proc. Natl. Acad. Sci. USA 103: 8459–8464. red blood cell transfusion. Vox Sang. 44: 212–217. 28. Alexopoulou, L., A. C. Holt, R. Medzhitov, and R. A. Flavell. 2001. Recognition 2. Fluit, C. R., V. A. Kunst, and A. M. Drenthe-Schonk. 1990. Incidence of red cell of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. antibodies after multiple blood transfusion. Transfusion 30: 532–535. Nature 413: 732–738. 3. Heddle, N. M., R. L. Soutar, P. L. O’Hoski, J. Singer, J. A. McBride, M. A. Ali, 29. Salem, M. L., S. A. El-Naggar, A. Kadima, W. E. Gillanders, and D. J. Cole. 2006. and J. G. Kelton. 1995. A prospective study to determine the frequency and The adjuvant effects of the toll-like receptor 3 ligand polyinosinic-cytidylic acid clinical significance of alloimmunization post-transfusion. Br. J. Haematol. 91: poly (I:C) on antigen-specific CD8+ T cell responses are partially dependent on NK 1000–1005. cells with the induction of a beneficial cytokine milieu. Vaccine 24: 5119–5132. 1050 IFN-a/b REGULATES RBC ALLOIMMUNIZATION

30. Meylan, E., and J. Tschopp. 2006. Toll-like receptors and RNA helicases: two 50. Sun, Q., L. Sun, H. H. Liu, X. Chen, R. B. Seth, J. Forman, and Z. J. Chen. 2006. parallel ways to trigger antiviral responses. Mol. Cell 22: 561–569. The specific and essential role of MAVS in antiviral innate immune responses. 31. McNab, F., K. Mayer-Barber, A. Sher, A. Wack, and A. O’Garra. 2015. Type I Immunity 24: 633–642. in infectious disease. Nat. Rev. Immunol. 15: 87–103. 51. Sato, M., H. Suemori, N. Hata, M. Asagiri, K. Ogasawara, K. Nakao, T. Nakaya, 32. Proietti, E., L. Bracci, S. Puzelli, T. Di Pucchio, P. Sestili, E. De Vincenzi, M. Katsuki, S. Noguchi, N. Tanaka, and T. Taniguchi. 2000. Distinct and es- M. Venditti, I. Capone, I. Seif, E. De Maeyer, et al. 2002. Type I IFN as a natural sential roles of transcription factors IRF-3 and IRF-7 in response to viruses for adjuvant for a protective immune response: lessons from the influenza vaccine IFN-alpha/beta gene induction. Immunity 13: 539–548. model. J. Immunol. 169: 375–383. 52. Honda, K., H. Yanai, H. Negishi, M. Asagiri, M. Sato, T. Mizutani, N. Shimada, 33. Le Bon, A., C. Thompson, E. Kamphuis, V. Durand, C. Rossmann, U. Kalinke, Y. Ohba, A. Takaoka, N. Yoshida, and T. Taniguchi. 2005. IRF-7 is the master and D. F. Tough. 2006. Cutting edge: enhancement of antibody responses regulator of type-I -dependent immune responses. Nature 434: 772– through direct stimulation of B and T cells by type I IFN. J. Immunol. 176: 777. 2074–2078. 53. Yamamoto, M., S. Sato, H. Hemmi, K. Hoshino, T. Kaisho, H. Sanjo, 34. Olsen, N., T. Sokka, C. L. Seehorn, B. Kraft, K. Maas, J. Moore, and T. M. Aune. O. Takeuchi, M. Sugiyama, M. Okabe, K. Takeda, and S. Akira. 2003. Role of 2004. A gene expression signature for recent onset rheumatoid arthritis in pe- adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. ripheral blood mononuclear cells. Ann. Rheum. Dis. 63: 1387–1392. Science 301: 640–643. 35. Higgs, B. W., Z. Liu, B. White, W. Zhu, W. I. White, C. Morehouse, P. Brohawn, 54. Adachi, O., T. Kawai, K. Takeda, M. Matsumoto, H. Tsutsui, M. Sakagami, P. A. Kiener, L. Richman, D. Fiorentino, et al. 2011. Patients with systemic lupus K. Nakanishi, and S. Akira. 1998. Targeted disruption of the MyD88 gene results erythematosus, myositis, rheumatoid arthritis and scleroderma share activation in loss of IL-1- and IL-18-mediated function. Immunity 9: 143–150. of a common type I interferon pathway. Ann. Rheum. Dis. 70: 2029–2036. 55. Smith, N. H., K. L. Henry, C. M. Cadwell, A. Bennett, J. E. Hendrickson, 36. Bave,˚ U., G. Nordmark, T. Lo¨vgren, J. Ro¨nnelid, S. Cajander, M. L. Eloranta, T. Frame, and J. C. Zimring. 2012. Generation of transgenic mice with anti- G. V. Alm, and L. Ro¨nnblom. 2005. Activation of the type I interferon system in thetical KEL1 and KEL2 human blood group antigens on red blood cells. primary Sjo¨gren’s syndrome: a possible etiopathogenic mechanism. Arthritis Transfusion 52: 2620–2630. Rheum. 52: 1185–1195. 56. Desmarets, M., C. M. Cadwell, K. R. Peterson, R. Neades, and J. C. Zimring. 37. Baechler, E. C., J. W. Bauer, C. A. Slattery, W. A. Ortmann, K. J. Espe, 2009. Minor histocompatibility antigens on transfused leukoreduced units of red J. Novitzke, S. R. Ytterberg, P. K. Gregersen, T. W. Behrens, and A. M. Reed. blood cells induce bone marrow transplant rejection in a mouse model. Blood 2007. An interferon signature in the peripheral blood of dermatomyositis patients 114: 2315–2322. Downloaded from is associated with disease activity. Mol. Med. 13: 59–68. 57. Peterson, K. R., H. Fedosyuk, L. Zelenchuk, B. Nakamoto, E. Yannaki, 38. Assassi, S., M. D. Mayes, F. C. Arnett, P. Gourh, S. K. Agarwal, G. Stamatoyannopoulos, S. Ciciotte, L. L. Peters, L. M. Scott, and T. A. McNearney, D. Chaussabel, N. Oommen, M. Fischbach, K. R. Shah, et al. T. Papayannopoulou. 2004. Transgenic Cre expression mice for generation of 2010. Systemic sclerosis and lupus: points in an interferon-mediated continuum. erythroid-specific gene alterations. Genesis 39: 1–9. Arthritis Rheum. 62: 589–598. 58. Stowell, S. R., K. L. Henry, N. H. Smith, K. E. Hudson, G. R. Halverson, 39. Ytterberg, S. R., and T. J. Schnitzer. 1982. Serum interferon levels in patients J. C. Park, A. M. Bennett, K. R. Girard-Pierce, C. M. Arthur, S. T. Bunting, et al. with systemic lupus erythematosus. Arthritis Rheum. 25: 401–406. 2013. Alloantibodies to a paternally derived RBC KEL antigen lead to hemolytic

40. Feng, X., H. Wu, J. M. Grossman, P. Hanvivadhanakul, J. D. FitzGerald, disease of the fetus/newborn in a murine model. Blood 122: 1494–1504. http://www.jimmunol.org/ G. S. Park, X. Dong, W. Chen, M. H. Kim, H. H. Weng, et al. 2006. Association 59. Lund, J., A. Sato, S. Akira, R. Medzhitov, and A. Iwasaki. 2003. Toll-like re- of increased interferon-inducible gene expression with disease activity and lupus ceptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid den- nephritis in patients with systemic lupus erythematosus. Arthritis Rheum. 54: dritic cells. J. Exp. Med. 198: 513–520. 2951–2962. 60. Longhi, M. P., C. Trumpfheller, J. Idoyaga, M. Caskey, I. Matos, C. Kluger, 41. Crow, M. K., K. A. Kirou, and J. Wohlgemuth. 2003. Microarray analysis of A. M. Salazar, M. Colonna, and R. M. Steinman. 2009. Dendritic cells require a interferon-regulated genes in SLE. Autoimmunity 36: 481–490. systemic type I interferon response to mature and induce CD4+ Th1 immunity 42. Baechler, E. C., F. M. Batliwalla, G. Karypis, P. M. Gaffney, W. A. Ortmann, with poly IC as adjuvant. J. Exp. Med. 206: 1589–1602. K. J. Espe, K. B. Shark, W. J. Grande, K. M. Hughes, V. Kapur, et al. 2003. 61. Montoya, M., G. Schiavoni, F. Mattei, I. Gresser, F. Belardelli, P. Borrow, and Interferon-inducible gene expression signature in peripheral blood cells of pa- D. F. Tough. 2002. Type I interferons produced by dendritic cells promote their tients with severe lupus. Proc. Natl. Acad. Sci. USA 100: 2610–2615. phenotypic and functional activation. Blood 99: 3263–3271. 43. Bronson, P. G., C. Chaivorapol, W. Ortmann, T. W. Behrens, and R. R. Graham. 62. Ohyagi, H., N. Onai, T. Sato, S. Yotsumoto, J. Liu, H. Akiba, H. Yagita,

2012. The genetics of type I interferon in systemic lupus erythematosus. Curr. K. Atarashi, K. Honda, A. Roers, et al. 2013. Monocyte-derived dendritic cells by guest on September 23, 2021 Opin. Immunol. 24: 530–537. perform hemophagocytosis to fine-tune excessive immune responses. Immunity 44. Yao, Y., L. Richman, B. W. Higgs, C. A. Morehouse, M. de los Reyes, 39: 584–598. P. Brohawn, J. Zhang, B. White, A. J. Coyle, P. A. Kiener, and B. Jallal. 2009. 63. Calabro, S., A. Gallman, U. Gowthaman, D. Liu, P. Chen, J. Liu, Neutralization of interferon-alpha/beta-inducible genes and downstream effect in J. K. Krishnaswamy, M. S. Nascimento, L. Xu, S. R. Patel, et al. 2016. Bridging a phase I trial of an anti-interferon-alpha monoclonal antibody in systemic lupus channel dendritic cells induce immunity to transfused red blood cells. J. Exp. erythematosus. Arthritis Rheum. 60: 1785–1796. Med. 213: 887–896. 45. Petri, M., D. J. Wallace, A. Spindler, V. Chindalore, K. Kalunian, E. Mysler, 64. Gilson, C. R., and J. C. Zimring. 2012. Alloimmunization to transfused platelets C. M. Neuwelt, G. Robbie, W. I. White, B. W. Higgs, et al. 2013. , a requires priming of CD4+ T cells in the splenic microenvironment in a murine human anti-interferon-a monoclonal antibody, in systemic lupus erythematosus: model. Transfusion 52: 849–859. a phase I randomized, controlled, dose-escalation study. Arthritis Rheum. 65: 65. Scheu, S., P. Dresing, and R. M. Locksley. 2008. Visualization of IFNbeta 1011–1021. production by plasmacytoid versus conventional dendritic cells under specific 46. Stack, G., and C. A. Tormey. 2016. Estimating the immunogenicity of blood stimulation conditions in vivo. Proc. Natl. Acad. Sci. USA 105: 20416–20421. group antigens: a modified calculation that corrects for transfusion exposures. Br. 66. Patil, A., Y. Kumagai, K. C. Liang, Y. Suzuki, and K. Nakai. 2013. Linking J. Haematol. 175: 154–160. transcriptional changes over time in stimulated dendritic cells to identify gene 47. Noizat-Pirenne, F., C. Tournamille, P. Bierling, F. Roudot-Thoraval, P. Y. Le networks activated during the innate immune response. PLoS Comput. Biol. 9: Pennec, P. Rouger, and H. Ansart-Pirenne. 2006. Relative immunogenicity of e1003323. Fya and K antigens in a Caucasian population, based on HLA class II restriction 67. Edwards, A. D., S. S. Diebold, E. M. Slack, H. Tomizawa, H. Hemmi, T. Kaisho, analysis. Transfusion 46: 1328–1333. S. Akira, and C. Reis e Sousa. 2003. Toll-like receptor expression in murine DC 48. Marsh, W. L., and C. M. Redman. 1990. The kell blood group system: a review. subsets: lack of TLR7 expression by CD8 alpha+ DC correlates with unre- Transfusion 30: 158–167. sponsiveness to imidazoquinolines. Eur. J. Immunol. 33: 827–833. 49. Muller,€ U., U. Steinhoff, L. F. Reis, S. Hemmi, J. Pavlovic, R. M. Zinkernagel, 68. Smith, N. H., E. A. Hod, S. L. Spitalnik, J. C. Zimring, and J. E. Hendrickson. and M. Aguet. 1994. Functional role of type I and type II interferons in antiviral 2012. Transfusion in the absence of inflammation induces antigen-specific tol- defense. Science 264: 1918–1921. erance to murine RBCs. Blood 119: 1566–1569.