CD4+ T effector cells lymphocytes (Teff) are functionally heterogeneous, and traditionally divided into subsets defined by the cytokines they produce. To determine the actual states that Teff adopt in frontline tissues in vivo, we applied single-cell RNAseq on colonic Teff in a range of conditions: germfree, unchallenged Specific Pathogen Free, or after bacterial infection with Citrobacter rodentium or attenuated Salmonella typhimurium, or two helminths Heligmosomoides polygyrus and Nippostrongylus brasiliensis . We chose infection times that allowed responses to develop fully and achieve full Teff bias (day 11-13), and the pathogens elicited the expected biased cytokine responses. CD4+ cells from control... (continued)

Single cell transcriptomic analysis (InDrops) of mouse FoxP3- CD4+ Tconv cells and FoxP3+ regulatory CD4+ T cells isolated from secondary lymphoid organs.Abstract:CD4+ T regulatory cells (Treg) are central to immune homeostasis, their phenotypic heterogeneity reflecting the diverse environments and target cells that they regulate. To understand this heterogeneity, we combined single-cell RNA-seq, activation reporter and T cell receptor (TCR) analysis to profile thousands of Treg or conventional CD4+FoxP3- T cells (Tconv) from mouse lymphoid organs. Treg and Tconv pools showed areas of overlap, as resting 'furtive' Tregs (category 1) with overall similarity to Tconvs or as a convergence of activated... (continued)

Whole CD45+ splenocytes from B6 mice, 10X (Broad)As a first pass to develop common Standard Operating Protocols to produce compatible single-cell RNAseq across the consortium, Immgen labs (Wash U. St Louis, Broad, HMS) generated parallel datasets from spleens of un-perturbed C57Bl/6 mice.Per ImmGen SOP, mice were 5 w.o. C57Bl/6J (B6) mice bred at JAX, shipped one week prior and maintained in SPF conditions. Spleens were mechanically dissociated, and CD45+ cells were purified by flow cytometry (Aria) immediately prior to encapsulation on a 10X genomics instrument. Libraries were constructed with the Chromium Single Cell 3’ Library Kit. The libraries use a... (continued)

Whole CD45+ splenocytes from B6 mice, 10X (HMS)As a first pass to develop common Standard Operating Protocols to produce compatible single-cell RNAseq across the consortium, Immgen labs (Wash U. St Louis, Broad, HMS) generated parallel datasets from spleens of un-perturbed C57Bl/6 mice.Per ImmGen SOP, mice were 5 w.o. C57Bl/6J (B6) mice bred at JAX, shipped one week prior and maintained in SPF conditions. Spleens were mechanically dissociated, and CD45+ cells were purified by flow cytometry (Aria) immediately prior to encapsulation on a 10X genomics instrument. Libraries were constructed with the Chromium Single Cell 3’ Library Kit. The libraries use a... (continued)