TLR1- cluster of differentiation 281

Toll-like receptor 1 (TLR1) often designated as CD281 (cluster of differentiation 281), a member of the Toll-like receptor family recognizes pathogen-associated molecular pattern with specificity for gram-positive bacteria. TLR1 is a 786-residue type I transmembrane protein with a 581-amino acid leucine-rich extracellular domain (ECD), a 23-amino acid transmembrane domain (amino acids 582 to 604), and a 181-amino acid cytoplasmic Toll homology signalling domain (1, 2). TLR1 maps to chromosome 4p14 with a calculated molecular weight of 84 kDa (3, 4). TLR1 is most closely related to TLR6 and TLR10 with 68% and 48% overall amino acid sequence identity, respectively. Among members of the TLR family, TLR1 along with TLR6 comprise the most highly conserved pair and appear to have arisen more recently during evolution through a gene duplication event. Different length transcripts presumably resulting from use of alternative polyadenylation site, and/or from alternative splicing, have been noted for TLR4.

In vivo, two different sized transcripts for TLR1 are observed suggesting that the mRNA is alternatively spliced to generate two different forms of the protein. TLR1 mRNA is ubiquitously expressed and found at higher levels than the other TLRs. Of the major leukocyte populations, TLR1 is most highly expressed by monocytes, but is also expressed by macrophages, dendritic cells (DCs), polymorphonuclear leukocytes, B, T, and NK cells. While TLR1 expression is most significantly upregulated by autocrine IL-6, it is also elevated by IFN-γß, IL-10, and TNF-α. However, TLR1 level is unaffected by exposure to both Gram-positive and Gram-negative bacteria.

TLR1 along with TLR6 functions as a co-receptor for TLR2, which confers ligand specificity and enables cell signaling. Collectively, these receptor pairs mediate immune responses to a wide variety of acylated cell wall components derived from Gram-positive bacteria, Gram-negative bacteria, mycoplasma, spirochetes, and fungi. TLR1 also heterodimerizes with TLR4, not to enhance its function, but to inhibit TLR4 activity (5, 6). Defects in the TLR1/2 signaling pathway may account for human hyporesponsiveness to OspA vaccination.

Through the reciprocal exchange of extracellular domains between the human TLRs 1 and 6, it has been revealed that TLR1/2 and TLR2/6 receptor pairs exhibit different specificities toward many microbial agonists including diacylated and triacylated lipopeptides, which is determined by the region comprised of leucine-rich repeat motifs 9–12 of these receptors. A recent finding suggests that three nonsynonymous single nucleotide polymorphisms (SNPs) are located in this region of TLR1. A variant of TLR1 based upon the SNP P315L, located in the loop of LRR motif 11 (LRR11), is greatly impaired in mediating responses to lipopeptides. The P315L SNP may predispose certain individuals to infectious diseases for which the sensing of microbial cell components by TLR1 is critical to innate immune defense (7). Thus variation in the inflammatory response to bacterial lipopeptides is regulated by a common TLR1 transmembrane domain polymorphism that could potentially impact the innate immune response and clinical susceptibility to a wide spectrum of pathogens.

Reference:

1. Janeway, C. A., Jr, R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20: 197-216.
2. Takeda, K., T. Kaisho, S. Akira. 2003. Toll-like receptors. Annu. Rev. Immunol. 21: 335-376.
3.Beutler, B., Z. Jiang, P. Georgel, K. Crozat, B. Croker, S. Rutschmann, X. Du, K. Hoebe. 2006. Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large. Annu. Rev. Immunol. 24: 353-389.
4. Rock, F.L. et al. (1998) Proc. Natl. Acad. Sci. USA 95:588.
5. Ozinsky, A. et al. (2000) Proc. Natl. Acad. Sci. USA 97:13766.
6. Wyllie, D.H. et al. (2000) J. Immunol. 165:7125.
7. Katherine O. Omueti The Journal of Immunology, 2007, 178: 6387-6394.

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TLR10- cluster of differentiation 290

Toll-like receptor 10 (TLR10) often known as CD290 (cluster of differentiation 290), is the most recently identified human homolog of the Drosophila TOLL protein. Human TLR10 is an orphan member of the Toll-like receptor family that recognizes pathogen-associated molecular pattern. Like other members of the TLR family, TLR10 contains a signal peptide, multiple leucine-rich repeats, a cysteine-rich domain, a transmembrane domain, and a cytoplasmic TOLL interleukin-1 receptor domain. The human TLR10 gene occupies 3,269 bases arranged in three exons on the short arm of chromosome 4 (4p14) and encodes an 811-amino acid protein, approximately 50% identical to TLR1 and to TLR6. TLR10 is most closely related to TLR1 and TLR6 with 48% and 46% overall amino acid identity, respectively. Multiple alternatively spliced transcript variants encoding the same protein have been found for this gene (1).

In vivo, TLR10 mRNA expression is highest in immune system-related tissues including spleen, lymph node, thymus, tonsil. TLR10 mRNA is most highly expressed on B cells. In vitro, TLR10 is moderately upregulated by autocrine IFN-γ, IL-1β, IL-6, IL-10, and TNF-α in PMA-differentiated THP-1 cells. TLR10 mRNA expression in THP-1 cells is elevated after exposure to both Gram-positive and Gram-negative bacteria. Ex vivo, monocyte TLR10 expression increases while granulocyte expression decreases on exposure to Gram-negative bacteria. (2, 3)

Due to absent of its rat homologue, the natural ligand for TLR10 has not been identified yet. Genomic studies indicate that TLR10 is in the same locus that contains TLR1 and TLR6 and are also structurally similar to each other. It has been speculated that, like TLR1 and TLR6, TLR10 may form a heterodimer with TLR2 and thereby be sensitive to similar pathogen-associated molecular patterns (PAMPs). Recent studies have shown that TLR10 was not only able to homodimerize but also heterodimerize with TLRs 1 and 2. (1)

TLR10 has been identified as a potential asthma candidate gene because early life innate immune responses to ubiquitous inhaled allergens and PAMPs may influence asthma susceptibility and thus TLR10 genetic variation may often contributes to asthma risk. (4)

References:
1. The Journal of Immunology, 2005, 174: 2942-2950.
2. Chuang, T. & R.J. Ulevitch (2001) Biochim. Biophys. Acta 1518:157.
3. Zarember, K.A. & P.J. Godowski (2002) J. Immunol. 168:554.
4. Hornung, V. et al. (2002) J. Immunol. 168:4531.

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In Search of Putative FOXP3+ Cell Surface Markers

Despite intense interest and scrutiny focused on FOXP3 as a key protein & master transcription factor, isolating and enriching for viable FOXP3 positive cells remains a challenge. Although cell separation/staining via CD4+CD25+ selection is commonly used, this technique has limited applications. Thus, surface markers specific for FOXP3 positive cells would be invaluable research tools as they would:

• Facilitate isolation & purification of viable
Treg cells
• Distinguish naturally occurring
CD4+CD25+cells from both naive and
recently activated CD4+CD25-
nonregulatory T cells
• Allow therapeutic manipulation of
Treg cells

As the search for putative FOXP3+ markers continue, Neuropilin-1, GPR83, & FR4 have emerged as potential candidates. IMGENEX is excited to offer a panel of flow cytometric characterized antibodies against:

• Neuropilin-1 (clone 211H6.01)
• GPR83 (polyclonal)
• Folate Receptor 4 (clones 12A5 & TH6)

Flow cytometric analysis of intracellular FOXP3 (IMG-5802D) and cell surface FR4 with clone 12A5 (IMG-6217C) (left) and clone TH6 (IMG-6218C) (right) at 0.06 ug/10^6 mouse splenocytes.

Flow cytometric analysis of Neuropilin-1 in CD4+CD25+ human PBMCs using A) an isotype control & B) DDX0440 at 0.5 ug/10^6 cells. These antibodies are available in multiple sizes and conjugates.

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