T regulatory cell chemokine production mediates pathogenic T cell attraction and suppression
Scott J. Patterson, … , Constadina Panagiotopoulos, Megan K. Levings
Scott J. Patterson, … , Constadina Panagiotopoulos, Megan K. Levings
Published February 8, 2016
Citation Information: J Clin Invest. 2016;126(3):1039-1051. https://doi.org/10.1172/JCI83987.
View: Text | PDF
Research Article Immunology

T regulatory cell chemokine production mediates pathogenic T cell attraction and suppression

Abstract

T regulatory cells (Tregs) control immune homeostasis by preventing inappropriate responses to self and nonharmful foreign antigens. Tregs use multiple mechanisms to control immune responses, all of which require these cells to be near their targets of suppression; however, it is not known how Treg-to-target proximity is controlled. Here, we found that Tregs attract CD4+ and CD8+ T cells by producing chemokines. Specifically, Tregs produced both CCL3 and CCL4 in response to stimulation, and production of these chemokines was critical for migration of target T cells, as Tregs from Ccl3–/– mice, which are also deficient for CCL4 production, did not promote migration. Moreover, CCR5 expression by target T cells was required for migration of these cells to supernatants conditioned by Tregs. Tregs deficient for expression of CCL3 and CCL4 were impaired in their ability to suppress experimental autoimmune encephalomyelitis or islet allograft rejection in murine models. Moreover, Tregs from subjects with established type 1 diabetes were impaired in their ability to produce CCL3 and CCL4. Together, these results demonstrate a previously unappreciated facet of Treg function and suggest that chemokine secretion by Tregs is a fundamental aspect of their therapeutic effect in autoimmunity and transplantation.

Authors

Scott J. Patterson, Anne M. Pesenacker, Adele Y. Wang, Jana Gillies, Majid Mojibian, Kim Morishita, Rusung Tan, Timothy J. Kieffer, C. Bruce Verchere, Constadina Panagiotopoulos, Megan K. Levings

×

Figure 1

Mouse FOXP3+ Tregs produce CCL3 and CCL4.

Options: View larger image (or click on image) Download as PowerPoint
Mouse FOXP3+ Tregs produce CCL3 and CCL4. (A–C) CD4+ T cells were sorted...
(A–C) CD4+ T cells were sorted into CD4+FOXP3+EGFP+ Tregs and CD4+FOXP3–EGFP– Tconv cells and stimulated for 48 hours with plate-bound α-CD3 (10 μg/ml) and soluble α-CD28 (2.5 μg/ml) in the presence of IL-2 (100 U/ml). (A) Ccl3 and Ccl4 mRNAs were quantified by RT-PCR and normalized to 18S rRNA. Expression is shown relative to unstimulated ex vivo levels. Data are the average ± SEM from 3 independent experiments. (B) Amounts of CCL3 and CCL4 in supernatants were determined using CBA flex sets. Data are the average ± SEM from 3 independent experiments. (C) Amounts of CCL3, CCL4, and CCL5 in supernatants were determined as in B after stimulation with the indicated amount of plate-bound α-CD3 (1 or 10 μg/ml), together with soluble α-CD28 and IL-2 as in A and B. Data are the average ± SEM from 3 independent experiments. (D) CD4+FOXP3+ Tregs and CD4+FOXP3– Tconv cells were stimulated for 72 hours as in A and B, then restimulated with PMA/ionomycin. The proportions of CCL3+ or IFN-γ+ cells within the live FOXP3+ or FOXP3–CD4+ gate were determined; gates were set based on equivalently stained unstimulated cells. Flow cytometry plots depict a representative experiment, with the average ± SEM of 3 independent experiments on the right. (E) CD4+FOXP3EGFP– cells were placed under iTreg differentiation (coated α-CD3 10 mg/ml, α-CD28 2.5 mg/ml, TGF-β 10 ng/ml, and IL-2 100 U/ml) or neutral (coated α-CD3 10 mg/ml, α-CD28 2.5 mg/ml, and IL-2 100 U/ml) conditions for 3 days, then restimulated with PMA/ionomycin. The proportions of CCL3+ or IFN-γ+ cells within live FOXP3+ cells or neutral conditions were determined and compared with data from ex vivo FOXP3+ Tregs. Left panel depicts a representative experiment; right panel shows the average ± SEM of 3 independent experiments. (F) Jurkat T cells were transfected with a pGL3 CCL3 or CCL4 promoter reporter construct together with an empty vector or a FOXP3-expressing vector. Normalized luciferase activity of the reporter construct is shown for 3 (CCL3) and 4 (CCL4) independent experiments. *P < 0.05 by paired t test.