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Blood, Vol. 113, Issue 12, 2684-2694, March 19, 2009
Complement-dependent T-cell lymphopenia caused by thymocyte deletion of the membrane complement regulator Crry
Blood Miwa et al.
113: 2684
Supplemental materials for: Miwa et al
Methods RT-PCR of Crry cDNAs and transfection of Chinese hamster ovary (CHO) cells
Tissue RNAs were prepared using the TRIzol reagents (Invitrogen). First strand cDNA for RT-PCR were synthesized as previously described 6 using total RNAs from kidney, liver and thymus and oligo(dT) as a primer. Full-length and truncated form of the mouse Crry cDNA were amplified by RT-PCR with the following Crry-specific primers: P3, 5′-CTGCAGGTAAAACGTTGTTTGAGAACGGTG-3′ (5′ UTR of mouse Crry cDNA); P4A, 5′-GAATTCCGTGCTGGGCTAGTGGT-3′ (3′ UTR of mouse Crry cDNA). The cDNAs were first cloned into the pCR2.1 vector and then subcloned into the eukaryotic expression vector pCDNA3 (Invtrogen, Carlsbad, CA) at EcoRI site. CHO cells (ATCC, Rockville, MD) were cultured in F12 medium containing 10% fetal bovine serum (FBS). They were seeded at 95% confluency in 100 mm dishes and transfected with 24 µg of the appropriate plasmid DNA using Lipofectamine (GIBCO/Life Technologies, Grand Island, NY) by following the manufactures instruction. Two days after trasnfection, G-418 (GIBCO/BRL) was added to the cell culture medium at 700 µg/ml to select transfected cells. Expression of full-length or truncated form Crry on G-418 resistant cells were examined by fluorescence-activated cell sorting (FACS) analysis using a rabbit anti-mouse Crry polyclonal antibody. Retroviral transduction of bone marrow stem cells
Full-length or truncated form of Crry cDNA was subcloned at EcoRI site into MigR1, a biscistronic retroviral vector carrying a green fluorescence protein (GFP) marker downstream of EMCV IRES (encephalomyocarditis virus internal ribosomal entry site) in tandem with the transgene.3,4 The MigR1 retroviral vector was kindly provided by Dr. WS Pear (University of Pennsylvania, Philadelphia, PA)4 Production of viral particles encoding Crry and transduction of bone marrow stem cells of DAF−∕−Crry−∕−C3−∕− mice were carried out as described previously.7 Mice were used two months after BM transfer. Approximately 10–30% of peripheral erythrocytes in the recipient mice were found to be positive for GFP as a marker transgene. Preparation of Crry SCR3/4-speicifc antibodies
A rabbit anti-mouse Crry antibody (kindly provided by Dr John Lambris, University of Pennsylvania) was biotinylated according to previously described protocol.7 To prepare an antibody that recognizes the intact Crry protein but not the mutant form of Crry lacking SCR 3 and 4 (CrryΔ3–4), biotinylated rabbit anti-mouse Crry was absorbed 4 times against CHO cells stably transfected with the CrryΔ3–4 cDNA (0.5 ml antibody at 150 µg/ml mixed with 3 × 108 cells, gentle shaking at 4°C overnight). Western blotting
CHO cells or mouse tissues (kidney, thymus) were homogenized and solubilized in 50 mM Tris buffer containing 1 mM EDTA, 1% Nonidet P-40, 0.5% deoxcholic aicd (Sigma, St. Louis, MO), 0.1% SDS, 150 mM NaCl, and 1 × protease inhibitor cocktail (Sigma). Nuclei and cytoplasmic debris were pelleted at 14,000 rpm on a microcentrifuge for 10 min at 4C. SDS-PAGE was performed with 100 µg/lane (for cells) and 150µg∕lane (for tissues) of solubilized proteins under non-reducing conditions. Western blot was performed using intact or absorbed rabbit anti-mouse Crry polyAbs and a mouse anti–β-actin mAb (Sigma). Western blot detection was achieved with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (Amersham Pharmacia Biotech, Piscataway, NJ) or HRP-conjugated anti-mouse IgG (Sigma) and ECL-plus kit (Amersham Pharmacia Biotech). Flow cytometry
To stain CHO cells and erythrocytes, cells were first incubated with biotinylated rabbit anti-mouse Crry polyAbs in FACS buffer (0.1% BSA, 0.1% Azide in PBS). After washing 2 times with FACS buffer, they were stained with streptavidin-APC. Single cell suspensions were prepared from the thymus, spleen and lymph nodes. These cells and blood PBMC were pre-incubated with purified anti-CD16 (Fc-block) before staining for specific antigens. Cells (1.5 × 106 cells in 30 _L FACS buffer) were stained with the following antibodies: anti–DAF-PE, anti–CD4-PerCP, anti–CD8-PE, anti–B220-FITC, anti–CD3-FITC (all from Pharmingen, San Diego, CA), anti–C3-FITC (ICN, Orangeburg, NY), or biotinylated anti-Crry antibody and streptavidin-APC (Pharmingen). Flow cytometry data were acquired on a FACScalibur machine and analyzed with the FlowJo software (Tree Star). Results CrryΔ3–4 protein is non-functional when expressed on mouse erythrocytes
To assess if CrryΔ3–4 is functional, we expressed CrryΔ3–4 on erythrocytes of Crry−∕−/DAF−∕−C3−∕− mice by retroviral vector-mediated gene transduction of bone marrow (BM) stem cells. As alluded to earlier, global Crry−∕− knockout was lethal.1 However, mice deficient in both Crry and complement protein C3 (Crry−∕−C3−∕−), obtained from Crry+∕−C3+∕− breeding, were viable.1 We used Crry−∕−/DAF−∕−C3−∕− mice 2 for this experiment because the combined deficiency of endogenous Crry and DAF made their erythrocytes an ideal low background platform for assessing the complement-regulating activity of ectopically expressed CrryΔ3–4. We transduced BM cells of Crry−∕−/DAF−∕−C3−∕− mice with CrryΔ3–4 or full-length Crry cDNA using the bicistronic retroviral vector Migir-1 which contained green fluorescence protein (GFP) as a gene transduction marker.3,4 As shown in Fig. S1, non-manipulated WT and Crry−∕−/DAF−∕−C3−∕− mice contained no GFP+ erythrocytes, whereas Crry−∕−/DAF−∕−C3−∕− mice reconstituted with BM cells transduced with either Migir-1-Crry or Migir-1-CrryΔ3–4 had a population of GFP+ erythrocyte in their peripheral blood (Fig. S1A, left panels). Further FACS analysis using anti-Crry or anti-SCR3/4 confirmed the expression of full-length Crry or CrryΔ3–4 on GFP+ but not GFP− erythrocytes (Fig. S1A, middle and right panels). We next compared the sensitivity of GFP+ and GFP− erythrocytes within the same BM chimera mice to classical pathway complement activation. Cells were sensitized with anti-erythrocyte antibodies, and after exposure to mouse serum, were analyzed by FACS for C3 deposition.5 As shown in Fig. S1B, GFP+ erythrocytes in full-length Crry cDNA-transduced chimeras incurred much lower C3 deposition than GFP− cells, confirming the complement-regulating activity of Crry. In contrast, the level of C3 deposition on GFP+ erythrocytes was similar to that of GFP− erythrocytes in CrryΔ3–4 cDNA-transduced chimeras, suggesting that CrryΔ3–4 is non-functional as a complement regulator. CrryΔ3–4 protein expressed on thymocytes is also non-functional as a complement regulator
To further confirm that the truncated Crry protein is non-functional as a complement regulator, we compared thymocytes from Lck-Cre+-Crryflox∕flox mice and Crry−∕−/C3−∕− mice (complete Crry knockout) for their sensitivity to alternative pathway complement attack ex vivo. Because thymocytes in Lck-Cre+-Crryflox∕flox mice were already partially opsonized with C3 (Fig 4), we generated bone marrow chimeras by transplanting bone marrow cells from Lck-Cre+-Crryflox∕flox, Crry−∕−/C3−∕− and WT mice to C3−∕− mice to normalize the respective thymocytes. After 8 weeks, we harvested thymocytes from the three types of chimeras and treated them with 80% mouse serum in an alternative pathway complement activation assay. As shown in Fig. S2, compared with WT thymocytes, Crry-deficient thymocytes were highly sensitive to alternative pathway complement attack. Importantly, thymocytes with a truncated Crry protein were as sensitive as Crry-deficient thymocytes to complement attack. This result unequivocally established that the mutated Crry protein is inactive as a complement regulator. REFERENCES 1. Xu C, Mao D, Holers VM, Palanca B, Cheng AM, Molina H. A critical role for murine complement regulator crry in fetomaternal tolerance. Science. 2000;287:498–501.
2. Molina H, Miwa T, Zhou L, et al. Complement-mediated clearance of erythrocytes: mechanism and delineation of the regulatory roles of Crry and DAF. Decay-accelerating factor. Blood. 2002;100:4544–4549. Epub 2002 Aug 4541.
3. Pear WS, Aster JC, Scott ML, et al. Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles. J Exp Med. 1996;183:2283–2291.
4. Pear WS, Miller JP, Xu L, et al. Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. Blood. 1998;92:3780–3792.
5. Miwa T, Zhou L, Hilliard B, Molina H, Song WC. Crry, but not CD59 and DAF, is indispensable for murine erythrocyte protection in vivo from spontaneous complement attack. Blood. 2002;99:3707–3716.
6. Song WC, Deng C, Raszmann K, et al. Mouse decay-accelerating factor: selective and tissue-specific induction by estrogen of the gene encoding the glycosylphosphatidylinositol-anchored form. J Immunol. 1996;157:4166–4172.
7. Kim DD, Miwa T, Song WC. Retrovirus-mediated over-expression of decay-accelerating factor rescues Crry-deficient erythrocytes from acute alternative pathway complement attack. J Immunol. 2006;177:5558–5566.
Files in this Data Supplement:
- Figure S1. CrryΔ3–4 is functionally inactive as a complement regulator when expressed on erythrocytes (JPG, 124 KB)
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(A) Crry or CrryΔ3–4 expression on erythrocytes of WT and Crry−∕−/DAF−∕−/C3−∕− (triple KO) mice with (Migir-1) or without (control) retroviral transduction of bone marrow stem cells. The Migir-1 vector contained either full-length Crry or CrryΔ3–4. Green fluorescent protein (GFP) was used as a marker for positive retroviral gene transduction. Erythrocytes were stained with α-Crry or α-SCR3/4, and GFP-positive (+) or -negative cells (−) were gated and analyzed for WT or mutant Crry expression. (B) Erythrocytes from Migir-1 transduced Crry−∕−/DAF−∕−/C3−∕− mice were sensitized with anti-erythrocyte mAbs 39,40 and exposed to two dilutions (1:320 and 1:640) of mouse serum in GVB++ (assay buffer) or in GVB++-EDTA (negative control). Erythrocytes expressing the full-length Crry (+, GFP-positive) incurred less C3 deposition than the non-transfected cells (−, GFP-negative). In contrast, C3 deposition on erythrocytes expressing CrryΔ3–4 (GFP-positive) was similar to that on non-transfected cells (GFP-negative).
- Figure S2. CrryΔ3–4 on thymocytes was non-functional as an alternative pathway complement regulator (JPG, 64.1 KB)
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Thymocytes from WT→C3−∕−, Lck-Cre+-Crryflox∕flox →C3−∕− and Crry−∕−C3−∕−→C3−∕− chimeras were treated with 80% mouse serum ex vivo and analyzed for C3 deposition by FACS. WT thymocytes (thin line open histogram) were resistant to complement opsonization, whereas thymocytes from Lck-Cre+-Crryflox∕flox mice (Lck, filled histogram) with mutated Crry and that from Crry−∕−C3−∕− mice (Crry KO, thick line open histogram) lacking Crry displayed similar sensitivity to complement attack.
- Figure S3. Histology (H&E staining, A–C) and immunostaining of C3 (D–F) in Cre−-Crryflox∕flox, CD4-Cre+-Crryflox∕flox and Lck-Cre+- Crryflox∕flox mouse thymi (JPG, 120 KB)
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No structural abnormality was observed in CD4-Cre+-Crryflox∕flox and Lck-Cre+-Crryflox∕flox mouse thymi but numerous C3-positive cells were detected.
- Figure S4. CRIg deficiency failed to rescue the peripheral T-cell lymphpenia phenotype of Lck-Cre+-Crryflox∕flox mice (JPG, 147 KB)
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