IFN-γ and STAT1 are required for efficient induction of CXC chemokine receptor 3 (CXCR3) on CD4⁺ but not CD8⁺ T cells

To the editor:

The CXC chemokine receptor 3 (CXCR3) regulates migration and function of T cells during inflammation. Resting T cells express low levels of CXCR3 but they up-regulate CXCR3 upon activation. Several studies suggest that T helper 1 (Th1)/Th2-associated cytokines regulate levels of CXCR3 on T cells.^1–3  Interferon-γ (IFN-γ), which signals via STAT1, enhances CXCR3 expression on T cells,²  but its role in regulating CXCR3 on CD4⁺ versus CD8⁺ T cells is not clear. We therefore examined the role of IFN-γ and STAT1 in regulating CXCR3 levels on CD4⁺ and CD8⁺ T cells.

Naive CD4⁺ and CD8⁺ T cells from C57BL/6 mice were stimulated as described previously²  with anti-CD3e (3 μg/mL; Biolegend, San Diego, CA) and anti-CD28 (4 μg/mL; Biolegend) in the presence of anti–IFN-γ neutralizing or control antibody, and CXCR3 expression was analyzed by flow cytometry. CD4⁺ T cells activated in the presence of anti–IFN-γ Ab (clone no. XMG1.2; Pharmingen, San Diego, CA) expressed less CXCR3 than controls (Figure 1A). However, IFN-γ blockade had no effect on CXCR3 levels in CD8⁺ T cells (Figure 1B). Control CD4⁺ and CD8⁺ T cells up-regulated CXCR3 (Figure 1A,B) and produced comparable IFN-γ (data not shown).

Next, we compared CXCR3 surface expression and mRNA levels in stimulated CD4⁺ and CD8⁺ T cells from wild-type (WT) and STAT1^−/− C57BL/6 mice. Activated STAT1^−/− CD4⁺ T cells failed to up-regulate CXCR3 as efficiently as WT CD4⁺ T cells (Figure 1C). In contrast, STAT1^−/− CD8⁺ T cells showed a significant induction of CXCR3 similar to WT CD8⁺ T cells (Figure 1D). Low CXCR3 expression on STAT1^−/− CD4⁺ T cells also correlated with low CXCR3 mRNA levels (Figure 1E,F). Activated WT and STAT1^−/− CD4⁺ as well as CD8⁺ T cells produced significant IFN-γ, but levels were lower in STAT1^−/− T cells (WT CD4, 19908.17 [± 2582.868] pg/mL; WT CD8, 27361.05 [± 3484.116] pg/mL; STAT1^−/− CD4, 8012.928 [± 1627.75] pg/mL; STAT1^−/− CD8, 17152.14 [± 3680.819] pg/mL). Blockade of IFN-γ did not inhibit CXCR3 expression on STAT1^−/− CD8⁺ T cells, suggesting that IFN-γ was not inducing CXCR3 via a STAT1-independent pathway (data not shown).

We also determined whether the transcription factors T-bet and eomesodermin (Eomes) are involved in CXCR3 induction on STAT1^−/− CD8⁺ T cells by measuring mRNA levels by real-time reverse transcription–polymerase chain reaction (RT-PCR). Both these factors control CD4⁺ and CD8⁺ T-cell activity by regulating expression of many genes, including Cxcr3,⁴  and their expression in T cells can be induced via STAT1-depdendent and -independent mechanims.^5,6  STAT1^−/− CD4⁺ T cells showed less induction of T-bet mRNA than WT CD4⁺ T cells (Figure 1G). In contrast, STAT1^−/− CD8⁺ T cells contained more T-bet mRNA than WT CD8⁺ T cells, but the difference was not significant (Figure 1H). STAT1^−/− CD4⁺ and CD8⁺ T cells also contained less Eomes mRNA than WT T cells, but these differences were not significant (Figure 1I,J).

These findings show that IFN-γ and STAT1 are critical for efficient induction of CXCR3 on CD4⁺, but not CD8⁺ T cells upon activation. To best of our knowledge, this is the first study to demonstrate differential requirement for IFN-γ and STAT1 in up-regulation of CXCR3 on CD4⁺ versus CD8⁺ T cells. These findings are important in designing therapeutic strategies to control CXCR3 expression in T cells for treatment of autoimmune and inflammatory diseases.