Icyt reflection

Single cell suspensions were prepared from spleens and stained with fluorochrome-conjugated antibodies. For flow cytometry of the adoptive transfer and influenza experiments, Live/Dead Zombie Aqua, anti-CD45.1-AF700 (A20), anti-CD45.2-BV421 (104), anti-CD19-BV785 (6D5), and anti-CD23 biotin (B3B4), and anti-CD11c (N418) were from Biolegend. Anti-CD43-PE (S7) was from BD Biosciences. Cells were analyzed on an LSRII, and data analyzed using FlowJo software (Tree Star). Intracellular stains for T-bet were performed with anti-T-bet-APC (4B10) from Biolegend and the Foxp3 transcription factor kit (eBioscience) according to manufacturer's instructions. For FACS sorting to isolate subsets, anti-CD43-APC (S7) was from BD Biosciences. Anti-CD23-PE Cy7 (B3B4), anti-CD21/CD35-eFluor 450 (4E3), anti-CD45R-FITC (B220, RA3-6B2), and anti-CD93 (AA4.1)-APC were from eBioscience. Stained splenocytes were analyzed with a BD FACSCanto II, or sorted using a BD FACSAria III, BD FACSAria Fusion, iCyt Reflection (Sony Biotechnology), or Beckman Coulter MoFlo. Follicular (FO) B cells were isolated as CD93 (AA4.1)^- CD43^- B220⁺ CD21/35⁺ CD23⁺. Marginal zone (MZ) B cells were isolated as CD93 (AA4.1)^- CD43^- B220⁺ CD21/35⁺ CD23^Lo. ABCs were isolated as CD93 (AA4.1)^- CD43^- B220⁺ CD21/35^- CD23^-. Analyses were done using FlowJo software.

MLN T cells [Fixable viability dye (FVD)⁻CD90.2⁺CD3⁺] from treated C3H.SW recipients mice receiving allo-HCT were sorted for nanostring analysis. Total T cells (FVD⁻CD45⁺TCR-β⁺), CD4⁺ T cells, CD8⁺ T cells, CD45⁺TCR-β⁻ cells, stromal cells (FVD⁻CD45⁻Epcam⁻), epithelial cells (FVD⁻CD45⁻Epcam⁺), and endothelial cells (FVD⁻CD45⁻Epcam⁻CD146⁺) were sorted from single cell suspensions of intestine from treated C3H.SW recipient mice for quantitative reverse-transcription polymerase chain reaction (RT-PCR) or western blot analysis. Cell sorting was performed on BD FACSAria (BD Bioscience) or iCyt Reflection (Sony Biotechnology) in the flow cytometry core facility at Indiana University School of Medicine.

T cells were purified from spleen and lymph nodes of mice by FAC sorting with antibodies specific for CD4 (RM4–5), CD8 (53–6.7), CD25 (PC61.5), CD44 (IM7), CD45RB (C363–16A), and CD62L (MEL-14). Naive T cells were either CD25⁻CD44^lo or CD25CD44^loCD62L^hi. Regulatory T cells were CD4⁺CD25⁺CD45RB^lo. Cell sorting was performed using the iCyt Reflection or SY3200 Cell Sorters (Sony Biotechnology). Purity of sorted cells was confirmed via flow cytometry and greater than 94% in all experiments.

CD27⁺ or CD27⁻ fractions of YFP⁺ cells were sorted after enrichment with CD4 beads (Miltenyi) from spleens and lymph nodes of MOG-immunized WT or Rptor^Il17aCre mice. Sorting was performed on a Reflection (iCyt/Sony) cytometer, and sort-purified cells were transferred via retroorbital injection into Rag1^−/− mice. Due to low cell numbers, we used day 1 after transfer as the baseline reading to normalize subsequent analyses at days 14–15, as previously described^{27 (link)}, for donor cell abundance in the spleen and lymph nodes. Similar transfer experiments were performed using CD45.1⁺ hosts, except that 2D2-transgenic mice (expressing MOG antigen-specific T cell receptors^{28 (link)}; crossed onto the Il17aCre (R26R^eYFP) fate-mapping system) were used as donor mice, and CD4 T cell enrichment was performed at the time of analysis, to facilitate flow cytometric detection of the rare population of donor cells.