Featuring telomere-independent senescence, TEMRA cells especially build up in older human patients with persistent viral infections or inflammatory diseases [9,31]. the annealing and extension occasions for the PCR. The producing slope of the regression analysis corresponding to the efficiency of the qPCR as well as the dynamic range for detecting 100% positive of the lowest dilution are indicated. Calibration curve, melt curve and amplification plot for CD62L and CX3CR1 are depicted. 1297-9716-44-18-S1.pdf (205K) GUID:?78A8F562-35BC-4F2A-8D7D-9D983076D0D4 Additional file 2 Time course study of IFN-, TNF- and IL-2 production in CD27-sorted T helper cells. Supernatants of FACS-sorted Etidronate (Didronel) and ConA/rhIL-2 stimulated CD4+CD8-CD27+ (na?ve), CD4+CD8+CD27+ (CD27+) and CD4+CD8+CD27- (CD27-) cells were collected on day 1, 2 and 4 for ELISAs. The graphs show duration of cultivation on x-axes and mean values for the respective cytokine of duplicate wells in ng/mL on y-axes. Results of one animal are depicted. 1297-9716-44-18-S2.pdf (42K) GUID:?F6A13AE0-66B9-4530-A6A8-B8CE68FAC353 Additional file 3 Binding region of anti-CCR7 mAb. Alignment of full length CCR7 amino acid sequences of human, cattle and pig using GeneDoc Version 2.7.000 [44]. Sequences are derived from Gene Lender by BLAST analyses and homologies are obtained from UniGene (NCBI). Based Etidronate (Didronel) on the human CCR7 sequence (“type”:”entrez-protein”,”attrs”:”text”:”P32248″,”term_id”:”1352335″,”term_text”:”P32248″P32248 [CCR7_HUMAN] examined, UniProtKB/Swiss-Prot), the transmission peptide (grey), the extracellular (yellow), and cytoplasmic (turquois) domains are highlighted. The reddish box indicates the binding region of anti-CCR7 mAb 3D12 (BD Biosciences). 1297-9716-44-18-S3.pdf (28K) GUID:?7D1AD2D6-7540-4CDA-842A-743E8B350E8A Additional file 4 CD45RC expression on thymocytes. CD45RC expression (histograms) was analysed within four different subpopulations: CD4-CD8-, CD4+CD8+, CD4-CD8+ and CD4+CD8- thymocytes (gates shown on contour plot) by FCM including a live/death discrimination dye. Data of one representative animal out of six is usually shown. At least 1??105 cells per sample were acquired. 1297-9716-44-18-S4.pdf (51K) GUID:?05DDB985-7621-412D-9058-B20C8DF14B03 Abstract Differentiation of porcine T helper cells is still poorly investigated, partly due to a lack of monoclonal antibodies (mAbs) specific for molecules involved in this process. Recently, we recognized a mAb specific for porcine CD27 and showed that CD27 is expressed by all na?ve CD8- T helper cells but divides CD8+ T helper cells into a CD27+ and a CD27- subset. In the present study, detailed phenotypical and functional analyses of these T-helper cell subpopulations were Rabbit polyclonal to ACTR1A performed. Na?ve CD8-CD27+ T helper cells predominantly resided in various lymph nodes, whereas higher proportions of CD8+CD27+ and CD8+CD27- T helper cells were found in blood, spleen and liver. Both CD8+CD27+ Etidronate (Didronel) and CD8+CD27- T helper cells were capable of generating IFN- upon in vitro polyclonal stimulation and antigen-specific restimulation. Experiments with sorted CD8-CD27+, CD8+CD27+ and CD8+CD27- T-helper cell subsets following polyclonal stimulation revealed the lowest proliferative response but the highest ability for IFN- and TNF- production in the CD8+CD27- subset. Therefore, these cells resembled terminally differentiated effector memory cells as explained in human. This was supported by analyses of CCR7 and CD62L expression. CD8+CD27- T helper cells were mostly CCR7- and experienced considerably reduced CD62L mRNA levels. In contrast, expression of both homing-receptors was increased on CD8+CD27+ T helper cells, which also experienced a proliferation rate much like na?ve CD8-CD27+ T helper cells and showed intermediate levels of cytokine production. Therefore, much like human, CD8+CD27+ T helper cells displayed a phenotype and functional properties of central memory cells. Introduction A peculiarity of porcine T helper cells is the expression of CD8 on a substantial proportion of these cells in blood and secondary lymphatic organs [1,2]. In vitro stimulation by superantigens or mixed leukocyte reactions causes an up-regulation of CD8 expression on porcine T helper cells [1,3], and it was reported that CD8+ T helper cells proliferate in response to stimulation with recall antigen [4-6]. Therefore, CD8 expression is perceived as a marker for activated and memory T helper cells, whereas a CD4+CD8- phenotype is considered to define na?ve T helper cells [3]. In addition to CD8, the expression of CD45RC and swine leukocyte antigen-DR (SLA-DR) was investigated in previous studies to identify different memory stages of CD8+ T helper cells. Differentiation from na?ve CD8- to memory CD8+ T helper cells was described to be accompanied by a loss of CD45RC and an increase in SLA-DR expression [3]. However, Etidronate (Didronel) an accurate discrimination of functionally unique T helper cells following antigen contact has remained unsuccessful so far [7]. In human and mouse, differentiation of T helper cells is commonly defined by i) the expression of receptors for lymph node homing, ii) the expression of co-stimulatory molecules and iii) the capability to produce certain cytokines. With regard to.