Immune Netw.  2009 Aug;9(4):127-132. 10.4110/in.2009.9.4.127.

Expression and Function of TLR2 on CD4 Versus CD8 T Cells

• Lee SM
• Joo YD
• Seo SK

• KMID: 1474584
• DOI: http://doi.org/10.4110/in.2009.9.4.127

Abstract

BACKGROUND
Toll-like receptors (TLRs) play a fundamental role in innate immunity through their capacity to recognize pathogen-associated molecular patterns. Also, TLRs that are expressed in T cells are reported to function as co-stimulatory receptors. However, the functional capacity of TLRs on CD4 T and CD8 T cells has not been directly compared. Here we compared CD4 and CD8 T cell responses to TLR2 ligand plus TCR-mediated stimulation. METHODS: TLR2 expression was analyzed on T cell subsets under naive and alloantigen-primed conditions. We analyzed the effects of TLR2 co-stimulation on proliferation and survival of T cell subsets in vitro when stimulated with soluble anti-CD3 in the presence or absence of synthetic ligand Pam3CSK4. RESULTS: TLR2 expression on CD8 T cells was induced following activation; this expression was much higher than on CD4 T cells. Thus, the molecule was constitutively expressed on Listeria-specific memory CD8 T cells. Based on these expression levels, proliferation and survival were markedly elevated in CD8 T cells in response to the TLR2 co-stimulation by Pam3CSK4 compared with those in CD4 T cells. CONCLUSION: Our data show that TLR2 co-stimulation is more responsible for proliferation and survival of CD8 T cells than for that of CD4 T cells.

Keyword

TLR2; T cell co-stimulation; CD4 T cell; CD8 T cell

MeSH Terms

Immunity, Innate
Memory
T-Lymphocyte Subsets
T-Lymphocytes
Toll-Like Receptors
Toll-Like Receptors

Figure

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  Figure 1 Expression of TLR2 on CD4 and CD8 T cells. (A) T cells were isolated from spleen and lymph nodes of naïve B6 mice as described in Materials and Methods and stained with PE-Cy5-anti-CD4 or -CD8 and PE-anti-TLR2, and then analyzed by flow cytometry. The cells were adoptively transferred into lethally irradiated (850 cGy) Balb/c recipient. Recipient splenocytes were isolated on day 4 and stained with mAbs against H-2d, TLR2, and CD4 or CD8. The histogram for TLR2 expression was gated on H-2b+CD4+ or H-2b+CD8+ cells. (B) Balb/c mice were infected with L. monocytogenes and reinfected on day 25 p. i. with 5000 CFU. Splenocytes were isolated on day 5, stained with FITC-anti-CD8 and PE-LLO91-99 pentamer, and analyzed by flow cytometry.

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  Figure 2 TLR2 ligand co-stimulates T cell proliferation in the total T, CD4, and CD8 T cells. Total T cells were isolated as described in Materials and Methods and incubated with anti-CD3s in the presence or absence of TLR2 ligand Pam3CSK4 (PAM) for 64 h. (A) The T cell proliferation was determined via [3H]thymidine incorporation. Values are means and standard deviations of data from three independent experiments. **p<0.001, anti-CD3 vs. anti-CD3+PAM. (B) Cultured cells were treated with BrdU (2 µg) for the last 1 h and stained with PE-anti-CD4 or -CD8 mAb. The cells were then stained for incorporated BrdU and analyzed by flow cytometry. The histogram for BrdU incorporation was gated on CD4+ or CD8+ cells.

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  Figure 3 Effect of TLR2 co-stimulation on proliferation of CD4 T cell vs. CD8 T cells. CD4 and CD8 T cells were isolated as described in Materials and Methods and incubated with anti-CD3s in the presence or absence of TLR2 ligand PAM for 64 h. The cultured cells were treated with BrdU (2 µg) for the last 1 h, stained for incorporated BrdU, and analyzed by flow cytometry.

• 

  Figure 4 Effect of TLR2 co-stimulation on the survival of CD4 vs. CD8 T cells. Isolated CD4 (A) and CD8 T cells (B) were incubated with anti-CD3s in the presence or absence of TLR2 ligand PAM for 64 h. The cells were harvested, stained with FITC-Annexin-V and 7-AAD, and then analyzed by flow cytometry. %Survival was determined on annexin V-7-AAD-cells. Values are means and standard deviations of data from three independent experiments. *p<0.05, **p<0.001, anti-CD3 vs. anti-CD3+PAM. (C) Cells were harvested and stained for intracellular Bcl-xL or Bcl-2.

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  Figure 5 Specificity of TLR2 co-stimulation. CD4 and CD8 T cells were isolated from normal or TLR2-/- mice and incubated with anti-CD3s in the presence or absence of TLR2 ligand PAM for 64 h. Other cells were pretreated with purified anti-TLR2 mAb (2 µg/ml) before the stimulation. (A) Cultured cells were treated with BrdU (2 µg) for the last 1 h, stained for incorporated BrdU, and then analyzed by flow cytometry. (B) Cultured cells were treated with brefeldin A for the last 4 h and stained for intracellular IFN-γ.

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