Cd45.2 clone 104

Single-cell suspensions of BM were prepared by filtering crushed, pooled femurs, tibiae, and iliac crests from each mouse. BM mononuclear cells (MNCs) were isolated by Ficoll-Paque (GE Healthcare Life Sciences) density centrifugation and stained with a combination of fluorochrome-conjugated antibodies from eBioscience, BD Biosciences, or BioLegend: CD45.2 (clone 104), c-Kit (clone 2B8), Sca-1 (clone 108129), CD150 (clone TC15-12F12.2), CD48 (clone HM48-1), FLT3 (Clone A2F10), CD34 (clone RAM34), FcγR (clone 2.4G2), mature lineage (Lin) marker mix (B220 (clone RA3-6B2), CD11b (clone M1/70), CD4 (clone RM4-5), CD8a (clone 53-6.7), Ter-119 (clone Ter-119), Gr-1 (clone RB6-8C5)), and the viability stain propidium iodide (PI). Stained cells were either analyzed or sorted using a FACSAria with Diva software (BD Biosciences) based on the following surface marker profiles: LSK (Lin⁻ Sca-1⁺ c-Kit⁺), LT-HSC (Lin⁻ Sca-1⁺ c-Kit⁺ Flt3⁻ CD150⁺ CD48⁻), ST-HSC (Lin⁻ Sca-1⁺ c-Kit⁺ Flt3⁻ CD150⁻ CD48⁻), MPP2 (Lin⁻ Sca-1⁺ c-Kit⁺ Flt3⁻ CD150⁺ CD48⁺), MPP3 (Lin⁻ Sca-1⁺ c-Kit⁺ Flt3⁻ CD150⁻ CD48⁺), MPP4 (Lin⁻ Sca-1⁺ c-Kit⁺ Flt3⁺), myeloid-restricted progenitors (MyPro; Lin⁻ Sca-1⁻ c-Kit⁺), GMP (Lin⁻ Sca-1^- c-Kit⁺ CD150⁻ CD34⁺ FcγR⁺), CMP (Lin⁻ Sca-1⁻ c-Kit⁺ CD150⁻ CD34⁺ FcγR^lo) and MEP (Lin⁻ Sca-1⁻ c-Kit⁺ CD150⁻ CD34⁻ FcγR⁻). All flow cytometry data was analyzed using FlowJo software.

Bone marrow cells were isolated from FcγRIIB^–/–YaaIRF5^+/+ and FcγRIIB^–/–YaaIRF5^–/– donors and washed once in PBS followed by RBC lysis using RBC lysing buffer (MilliporeSigma, R7757). Donor marrow cells (10⁶ cells per mouse) were injected into the tail veins of B6.SJL-Ptprc^a Pepc^b/BoyJ (CD45.1) recipient mice after cesium irradiation of recipient mice with 2 doses of 500 rad separated by 3 to 4 hours. Recipient mice were subsequently placed in irradiated cages, and water was supplemented with sulfamethoxazole/trimethoprim (Hi-Tech Pharmaceuticals) (at 2/0.4 mg/mL). Immune cell engraftment was subsequently confirmed using flow cytometry of PBMCs and antibodies specific for CD45.1 (clone A20; BioLegend) and CD45.2 (clone 104; BioLegend).

MHCII+ dependent CD4+ T cell cytotoxicity assay was modified from that previously described [23 (link)]. RBC-lysed CD45.1 donor splenocytes were labeled with 0.2μM or 2μM CFSE in PBS for 10 minutes at 37°C, then washed twice with RPMI1640/20% FBS. CFSE^hi splenocytes were labeled with 10μM ID93 CD4 peptides at 37°C for 1 hour, then washed. CFSE^lo and CFSE^hi donor CD45.1 cells were combined 1:1 in PBS, then up to 10⁷ total injected i.v. into immunized CD45.2 recipient mice one week post-immunization, and controls one week post-saline. The next day, splenocytes were harvested from recipients, RBC-lysed, and stained for CD45.1 (clone A20, catalog # 110724), CD45.2 (clone 104, catalog # 109835) and MHC II (I-A^b clone AF6-120.1, catalog # 116416) (all BioLegend). Cell gating hierarchy was singlets > lymphocytes > CD45.1⁺ > MHC II⁺ > CFSE^hi or CFSE^lo. The specific lysis was calculated as described previously [24 (link)]:
$[1\mathrm{‐}(\mathrm{C}\mathrm{F}\mathrm{S}{\mathrm{E}}^{\mathrm{h}\mathrm{i}}{}_{\mathrm{i}\mathrm{m}\mathrm{m}\mathrm{u}\mathrm{n}\mathrm{i}\mathrm{z}\mathrm{e}\mathrm{d}}/\mathrm{C}\mathrm{F}\mathrm{S}{\mathrm{E}}^{\mathrm{l}\mathrm{o}}{}_{\mathrm{i}\mathrm{m}\mathrm{m}\mathrm{u}\mathrm{n}\mathrm{i}\mathrm{z}\mathrm{e}\mathrm{d}})/\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e}(\mathrm{C}\mathrm{F}\mathrm{S}{\mathrm{E}}^{\mathrm{h}\mathrm{i}}{}_{\mathrm{u}\mathrm{n}\mathrm{i}\mathrm{m}\mathrm{m}\mathrm{u}\mathrm{n}\mathrm{i}\mathrm{z}\mathrm{e}\mathrm{d}}/\mathrm{C}\mathrm{F}\mathrm{S}{\mathrm{E}}^{\mathrm{l}\mathrm{o}}{}_{\mathrm{u}\mathrm{n}\mathrm{i}\mathrm{m}\mathrm{m}\mathrm{u}\mathrm{n}\mathrm{i}\mathrm{z}\mathrm{e}\mathrm{d}})]\mathrm{*}100.$

Cell suspensions prepared from grafts or renal LN were incubated in round-bottom 96-well plates in 200 μl RPMI containing 10% FCS plus 20 ng ml⁻¹ PMA and 1 μg ml⁻¹ Ionomycin at 37 °C/5% CO₂. After 1 h BD GolgiStop (0.7 μl ml⁻¹) was added, then cells were cultured for an additional 3 h. The cell surface was stained for expression of CD4 (clone RM4-5, 1:400), CD8 (clone 53-6.7, 1:200), CD45.1 (clone A20⁻, eBioscience, 1:100) and CD45.2 (Clone 104, BioLegend, 1:150) enabling identification of host cells (CD4⁺CD8⁻CD45.2⁺ or CD4⁻CD8⁺CD45.2⁺) and adoptively transferred OT-I cells (CD4⁻CD8⁺CD45.1⁺). Cells were then fixed and permeabilized using BD Cytofix/Cytoperm reagents before staining for IL-2 expression (clone JES6-5H4, 1:200) and analysis by flow cytometry.