Originally published online as doi:10.1189/jlb.1005616 on March 21, 2006
Published online before print March 21, 2006
(Journal of Leukocyte Biology. 2006;79:1268-1270.)
© 2006
by Society for Leukocyte Biology
LTA recognition by bovine 
T cells involves CD36
Kirk Lubick and
Mark A. Jutila1
Veterinary Molecular Biology, Montana State University, Bozeman
1Correspondence: Veterinary Molecular Biology, Montana State University, 960 Technology Blvd., Bozeman, MT 59718. E-mail: uvsmj{at}montana.edu
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ABSTRACT
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CD36 has recently been shown to facilitate monocyte Toll-like receptor 2 (TLR2) recognition of lipoteichoic acid (LTA), much like CD14 in TLR4 recognition of lipopolysaccharide. We previously found that bovine 
T cells express CD36 transcripts. Here, we tested whether bovine T cells express CD36 protein and if so, whether it functions in a manner similar to the monocyte molecule. CD36 transcripts and internal and cell surface protein could be detected in resting, sorted T cells. Phorbol 12-myristate 13-acetate (PMA)/ionomycin treatment increased CD36 transcript levels (detectable at 4 h) and protein expression (internal and cell surface). Increased surface antigen expression was detectable by 24 h and was maximal at 72 h following PMA/ionomycin stimulation. Anti-CD36 monoclonal antibody inhibited increased macrophage-inflammatory protein-1
gene expression in T cells activated by LTA. In conclusion, T cells express CD36, previously thought to be a myeloid and endothelial cell-restricted surface antigen, and it contributes to responses by these cells to microbial LTA.
Key Words: innate • scavenger receptor • Toll-like receptors • PAMP
T cells are the first T cells to develop, can be found in sites of entry into the body (epithelial cell-associated tissues, such as gut and pulmonary mucosa), accumulate during inflammation, and are thought to be involved in innate immune responses against a wide spectrum of pathogens [1
2
3
4
]. In epithelial cell-associated tissues and sites of inflammation, cells of the innate immune system, such as myeloid cells, epithelial cells, dendritic cells, and some specialized T cells, including T cells, can encounter invading microbes via recognition of pathogen-associated molecular patterns (PAMPs) [5
6
7
]. In T cells, PAMPs, such as crude lipopolysaccharide (LPS) preparations, induce selective expression of some chemokines, such as macrophage-inflammatory protein-1 (MIP-1) and MIP-1ß [7
]. In global gene expression analyses, we found that bovine T cells express transcripts for a number of different PAMP receptors, including scavenger receptors (such as CD36), Toll-like receptors (TLRs), and CD11b, among others [7
]. The importance of these receptors in PAMP responses by T cells has not been characterized.
The CD36 PAMP receptor is a member of the scavenger receptor family of leukocyte antigens. It is thought to be restricted to monocytes and endothelial cells and serves as a receptor for oxidized lipids, apoptotic cells, thrombospondin-1, and Plasmodium falciparum-parasitized erythrocytes [8
9
10
]. As mentioned above, CD36 has been shown to facilitate monocyte TLR2 responses against lipoteichoic acid (LTA), analogous to the role CD14 plays in facilitating TLR4 recognition of LPS [10
]. It is interesting that CD36 is quite restrictive in its effect on TLR2 function, enhancing TLR2/TLR6 ligand (LTA) but not TLR2/TLR1 ligand {the synthetic triacylated lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (PAM3CSK4) interactions [10
]. In this report, we tested the hypothesis that CD36 serves a similar function on T cells.
In agreement with our earlier study [7
], CD36 transcripts were detected in resting, sorted T cells (Fig. 1
). As also seen in our earlier study [7
], stimulation of T cells, in this case with PMA/ionomycin for 4 h, increased the level of detectable CD36 mRNA (Fig. 1)
. CD36 protein expression in bovine T cells was examined by fluorescence-activated cell sorter (FACS) and immunohistochemical analyses using an anti-human CD36 monoclonal antibody (mAb), which cross-reacts with bovine CD36 [9
]. Anti-CD36 mAb stained a fraction of resting peripheral blood T cells, which was repeated in cells from four different calves (Fig. 2A
). PMA/ionomycin treatment increased surface expression of CD36 on T cells, which was detectable at 24 h. After 72 h, nearly all T cells were CD36-positive following PMA/ionomycin stimulation (Fig. 2B)
. A few non- T cells also expressed CD36, but this fraction was highly variable between samples, and it is unclear at this time whether they were natural killer cells, a small subset of ß T cells, or even B cells (data not shown). Intracellular CD36 was examined by staining acetone-fixed cytospin preparations of sorted, resting, and 72 h PMA/ionomycin-stimulated T cells (72 h was chosen based on the FACS analyses shown in Fig. 2
). In contrast to the expression of CD36 on only a fraction of resting T cells detected by FACS (Fig. 2)
, most resting T cells in cytospin preparations were CD36-positive, suggesting they expressed CD36 within intracellular granules (Fig. 3
). Following PMA/ionomycin activation for 72 h, there was an obvious increase in staining, suggesting new protein synthesis and based on the FACS analysis (Fig. 2)
, translocation of the intracellular pools to the cell surface (Fig. 3)
. Western blot analysis showed the anti-CD36-reactive molecule on T cells to be the predicted molecular mass of CD36 (
80 kDa, data not shown). These results document that T cells express intracellular and cell surface forms of the CD36 protein and that activation leads to an increase of the cell surface molecule.
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Figure 2. CD36 surface expression increases on T cells following stimulation with PMA/ionomycin. Two-color flow cytometry was performed using standard protocols, as described previously [4
]. Briefly, cells were stained sequentially with 20 µg/ml anti-CD36 mAb, phycoerythrin (PE)-conjugated goat anti-mouse, 10% mouse serum, and fluorescein isothiocyanate-conjugated GD3.8 (anti-pan T cell). All FACS was done on a BD FACSCalibur using Cell Quest software. (A) CD36 expression on resting T cells from four different animals. (B) CD36 expression on T cells at time zero (B, Panel A) and 12 h (B, Panel B), 24 h (B, Panel C), 48 h (B, Panel D), and 72 h (B, Panel E) after PMA/ionomycin treatment in complete RPMI (10% fetal bovine serum, antibiotics, HEPES). (B, Panels F and G) PE second stage-only and isotype-matched primary mAb, plus PE second stage-negative controls, respectively. Quadrant markers were set based on background staining with second-stage reagent alone. Values reflect the percent of T cells in the upper right quadrant. (B) Results are representative of three individual experiments.
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Figure 3. T cells expressed intracellular and cell surface forms of the CD36 protein. T cells were purified by panning on E-selectin cDNA L-cell transfectants, as described previously [11
], which yielded a preparation of >85% pure T cells without surface antibody, as occurred with the MACS separation protocol described in Figure 1
. Cytospin slide preparations of resting or 72 h PMA/ionomycin-stimulated, purified T cells were prepared, blocked in PBS containing 5% goat serum, and stained with 20 µg/ml anti-CD36 or GD3.8 anti- T cell mAb, and primary mAb was detected by addition of goat anti-mouse peroxidase-conjugated secondary antibody and 3-amino-9-ethylcarbazole developing solution (TAGO, Inc., Fort Wayne, IN). Anti-CD36-stained resting T cells (A), anti-CD36 (B), and GD3.8-stained T cells (C) treated with PMA/ionomycin for 72 h and second stage-alone, negative control (D) are shown. Images were taken at 40x original magnification.
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In earlier studies, we found that bovine T cells increase transcript levels for MIP-1 following 4 h stimulation with PAMPs, such as peptidoglycan and LPS [7
] and LTA (unpublished observations). To test whether CD36 contributes to the recognition of LTA by T cells, as recently shown for monocytes [10
], we used the conditions of our earlier assays and tested the effect of anti-CD36 mAb versus an isotype-matched, negative control mAb. As seen before for other PAMPs [7
], T cells from different animals displayed variable responses to LTA, but in each case where LTA induced MIP-1 up-regulation, anti-CD36 mAb, but not the negative control mAb, blocked the effect (Fig. 4
and data not shown). The anti-CD36 mAb effect was specific to TLR2/TLR6 function in that responses to PAM3CSK4 (TLR2/TLR1 agonist) were not affected (Fig. 4)
.
Our results demonstrate for the first time that T cells express CD36 at the RNA, protein, and functional level. CD36 is expressed on resting T cells, but surface levels increase considerably following PMA/ionomycin treatment. Consistent with recent findings for monocytes, CD36 facilitates T cell responses to LTA [10
]. This interaction likely requires TLR2/TLR6, although this awaits formal demonstration. Additional studies are under way to test whether T cell CD36 also functions as a receptor for oxidized low-density lipoproteins and/or apoptotic cells, other activities attributed to the monocyte molecule [8
]. Expression of CD36 on T cells is consistent with and provides additional support to the role of these cells in innate immunity.
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ACKNOWLEDGEMENTS
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This project has been funded in whole or in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Department of Health and Human Services, under Contract No. HHSN266200400009/N01-AI40009 and supported by Initiative for Future Agricultural and Food Systems Grant No. 00-52100-9612 from the USDA Cooperative State Research, Education, and Extension Service, USDA National Research Initiative 03-352041370, and the Montana State Agriculture Experiment Station. Funding from the NIH COBRA program supported the FACS analyses in this project.
Received October 28, 2005;
revised December 21, 2005;
accepted January 12, 2006.
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