Silvia Bolland, Francisco Borrego, Alexandra Gil-Krzewska, Herbert C

Silvia Bolland, Francisco Borrego, Alexandra Gil-Krzewska, Herbert C. of apoptotic cells, or an exacerbated autoimmunity when combined with Fcreleased from these cells, and the recruitment of natural regulatory T cells.4, 5, 6, 7 Post AC engulfment, macrophages appear to be capable of fully digesting AC-associated antigens and limit their access to antigen presentation compartments, and therefore are inefficient at T-cell priming.8 In contrast, DC are highly efficient at processing and presentation of engulfed antigens, using either direct or cross-presentation pathways, which either anergizes or activates Rabbit Polyclonal to Actin-beta potentially self-reactive T cells, depending on the context of antigen presentation.9, 10, 11 Under homeostatic conditions, anti-inflammatory cytokines and natural regulatory T cells channel DC self-antigen presentation to induce regulatory T-cell differentiation from na?ve CD4 T-cell precursors, and to tolerize effector CD8 T cells.12, 13 With the impaired clearance of AC, subsequent necrosis leads to the release of pro-inflammatory molecules that can supply the co-stimulatory signals for self-reactive T-cell activation by DC.14, 15 AC display various eat-me’ signals on their surface that can be recognized by phagocytes.16, 17 The most well-known eat-me’ signal is phosphatidylserine (PS), which is translocated from the inner leaflet to the outer leaflet of the plasma membrane during apoptosis.16, 18, 19 Among the many receptors known to bind PS20, 21, 22, 23, 24, 25, 26 are members of the CD300 family of receptors, including human CD300a,27 mouse CD300f28, 29 and CD300b.30 The human CD300 receptors are type I transmembrane proteins with single IgV-like extracellular domains that are mainly expressed by myeloid cells.31, 32, 33 The orthologous mouse family has a variety of names, including CMRF-like molecules (CLM),31, 32, 34 but for simplicity in this report we use the human nomenclature for both species. Mouse CD300f (CLM-1) possesses both activating and inhibitory signaling potentials for regulation of AC engulfment upon PS recognition. CD300f deficiency predisposes C57BL/6 mice to develop autoimmune disease, as the lack of CD300f accelerates SLE-like disease development in mice normalized to those from or and Levatin was significantly increased in AC plus pristane-injected clearance of AC, the distribution of i.v.-injected CFSE-labeled AC in spleens was analyzed. At 15?min post AC injection, AC were predominately distributed in marginal zone areas in both CD300f-deficient and WT mice; at 30 and 60?min, there were more AC located in the white pulp of spleens from CD300f-deficient than WT mice (Figures 5i and j). The co-localization of AC with DC was also increased in the white pulp of spleens from CD300f-deficient compared with WT mice (Figures 5k and l). Consistently, Levatin more splenic DC from CD300f-deficient mice engulfed the i.v.-injected PKH67-labeled AC than those from WT mice (Figures 5m and n). The enhanced efferocytosis by CD300f-deficient DC suggested that more AC-associated antigens would be engulfed and available to be processed and presented for T-cell priming in transcript, quantitative real-time PCR was carried out with the SYBR Green PCR Master (Roche, Branchburg, NJ, USA) with the following primers 5-GTGCCGATATACCTCAGGCT-3 and 5-ATGCATCGGTTTCAACAAGA-3. The amount of transcript was calculated with the 2-delta CT method, where delta CT equals (CT splenic DC efferocytosis Thymocytes from C57BL/6 mice were labeled with PKH67-GL (Sigma-Aldrich) as per the manufacturer’s instructions, gamma-irradiated at 20?Gy, and incubated for 6?h at 37?C to generate ACs. Labeled AC (2 107) were then i.v. transferred into cross-presentation assays, em Cd300f /em +/+ or em Cd300f /em ?/? mice were injected i.v. with em /em -irradiated EG-7-OVA cells (5 106 per mouse). One day later, DC were purified from spleen using CD11c MicroBeads (Miltenyi Biotec), followed by co-culture with CFSE-labeled OT-I CD8+ T cells at 1?:?3 ratio for 3 days. The cells were stained with APC-labeled anti-V em /em Levatin 2 TCR and the CFSE dilution of the CD8+ T cells (gated on V em /em 2 TCR positive cells) was analyzed by flow cytometry. Statistical analysis Significance of the difference between groups was evaluated by two-tailed Student’s em t /em -test or two-way ANOVA. Alpha level was set to 0.05. Acknowledgments The study was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases. We thank Drs. Silvia Bolland, Francisco Borrego, Alexandra Gil-Krzewska, Herbert C. Morse III, Venkateswara Simhadri and Hongsheng Wang for critically reading our manuscript. We thank Dr. Joseph Brzostowski for the technical help with the microscopy. We thank.