We hypothesize that the WC1 molecules expressed by a T cell contribute to its pathogen responsiveness and that co-expression of multiple WC1 gene products that bind the same pathogen could result in increased avidity for the pathogen and amplify the signal in a dose-dependent manner (i.e., the more WC1s of the same or multiple types that bind the pathogen, the stronger the cellular activation signal). and other genes as indicated. (B) PCR products were gel-purified and cloned into pCR2.1 and subsequently analyzed with Sanger sequencing. Multiple sequence alignment using BioEdit shows nucleotide sequences of TaqMan assay-amplified WC1 genes from cDNA relative to the reference gene sequence found in Genbank (see Table ?Table11 for accession numbers). Image_2.TIF (1.6M) GUID:?11CA5A54-2AEF-4B63-A732-AC9C0D750BB0 Figure S3: Sorting strategy to obtain WC1+ T cell subpopulations for single cell cloning. (A) Single-positive WC1.1+ or WC1.2+ and (B) double positive WC1.1+/WC1.3+ T cells were flow cytometrically analyzed and gates applied. The three gated cell populations were then evaluated for their level of cell division dye and the efluor-670low cells (indicative of multiple cell EHT 5372 divisions) were collected as shown. This is representative of multiple flow cytometric sorts. Image_3.tiff (1.3M) GUID:?0E0EB546-65F0-4E54-9C5F-3D93285FD550 Figure S4: Representative clones with variable numbers of WC1 gene transcripts. Examples (from the 78 total clones) that had transcripts for one to five WC1 gene transcripts. If the mean was less than 2 and SE was at below EHT 5372 zero, the gene was not included in the tally of transcripts in Figures ?Figures55 and ?and66 or Table ?Table3.3. (A) WC1.1 cohort of T cell clones from monoclonal antibodies (mAb) BAG25A+/CACTB32A? sorted cells expanded using expansion strategy 3 (and IL-2) or (B) WC1.2 cohort of T cell clones from mAb BAG25A?/CACTB32A+ sorted cells expanded with IL-2 with or without IL-15 and IL-18 supplementation. Moles of transcripts for each clone (mean??SE) for WC1 and TRDC (hatched EHT 5372 bars) are shown. Image_4.tiff (75K) GUID:?8ADD44E8-C191-4E48-9000-F796F4B22261 Abstract T cells have broad reactivity and actively participate in protective TNFRSF4 immunity against tumors and infectious disease-causing organisms. In -high species such as ruminants and other artiodactyls many T cells bear the lineage-specific markers known as WC1. WC1 molecules are scavenger receptors coded for by a multigenic array and are closely related to SCART found on murine T cells and CD163 found on a variety of cells. We have previously shown that WC1 molecules are hybrid pattern recognition receptors thereby binding pathogens as well as signaling co-receptors for the T cell receptor. WC1+ T cells can be divided into two major subpopulations differentiated by the WC1 genes they express and the pathogens to which they respond. Therefore, we hypothesize that optimal T cell responses are contingent on pathogen binding to WC1 molecules, especially since we have shown that silencing WC1 results in an inability of T cells from primed animals to respond to the pathogen priming of cattle cells in the WC1.1+ subpopulation respond by proliferation and interferon- production to spp. in recall responses (6, 7) whereas cells in the WC1.2+ subpopulation respond to other pathogens such as following infection (8). When cattle are infected with virulent EHT 5372 strains of both WC1+ lineages are recruited to the granulomas in infected cattle (9) but only the WC1.1+ cells respond to the vaccine strain BCG (10). Following to both protein and non-protein antigens while WC1+ and CD8+ T cells respond to BCG-infected macrophages (9, 11). Adaptive-like memory T cells are not confined to the bovine model having been described for specific subpopulations of murine T cells (12, 13) and to be sensitized by (14) and (15) while in humans and non-human primates memory T cells responses to mycobacteria (16C18), influenza (19), and malaria (20) have been reported. The 13 WC1 molecules can be divided into 10 WC1.1-types and 3 WC1.2-types based on signature insertions or deletions of amino acids in their most membrane-distal SRCR domain known as the a1 domain (Figure S1 in Supplementary Material). The first sequenced WC1 genes (21) and therefore considered to be the archetypal WC1.1 [coded for by (22)] and WC1.2 molecules [coded for by (22)] differ in their binding to despite considerable sequence.