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CD28 and ITK signals regulate autoreactive T cell trafficking

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Abstract

Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the co-inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) develop fatal autoimmunity characterized by lymphocytic infiltration into nonlymphoid tissues. Here, we demonstrate that the CD28 co-stimulatory pathway regulates the trafficking of self-reactive Ctla4−/− T cells to tissues. Concurrent ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation but instead causes self-reactive Ctla4−/− T cells to accumulate in secondary lymphoid organs. Despite excessive spontaneous T cell activation and proliferation in lymphoid organs, Itk−/−; Ctla4−/− mice are otherwise healthy, mount antiviral immune responses and exhibit a long lifespan. We propose that ITK specifically licenses autoreactive T cells to enter tissues to mount destructive immune responses. Notably, ITK inhibitors mimic the null mutant phenotype and also prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of type 1 diabetes, highlighting their potential utility for the treatment of human autoimmune disorders.

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Figure 1: B7 signals regulate Ctla4−/− T cell migration.
Figure 2: ITK deficiency prolongs the lifespan of Ctla4−/− mice.
Figure 3: ITK deficiency prevents Ctla4−/− T cell infiltration into tissues.
Figure 4: Impaired transendothelial migration by DKO T cells.
Figure 5: ITK inhibitors moderate autoimmunity.

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Acknowledgements

We thank E. Huseby, B. Seed and R. Friedline for discussion, S. Turley for advice with stromal cells, T. Hunig (University of Wurzburg) for the SACD28 antibody, M. Coles for assistance with microscopy, M. Krummel for advice on imaging, D. Serreze for studies with diabetogenic CD8+ T cells, E. Huseby (University of Massachusetts Medical School) for MHC class II–deficient Rag1−/− mice and R. Welsh for the LCMV infection protocol. Core resources supported by the University of Massachusetts Medical School Diabetes Endocrinology Research Center grant DK32520 were used. This work was supported by US National Institutes of Health (NIH) grants to D.L.G. (AI46629, AI050864), S.L.S. (AI046530), L.J.B. (AI083505) and J.K. (RC1 DK086474 and AI083505). US NIH Chemical Genomics Center was supported by the Molecular Libraries Initiative and the Intramural Research Program of the NIH National Human Genome Research Institute.

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Contributions

N.J., L.J.B. and J.K. designed experiments, N.J. and B.M. performed experiments and analyzed data, K.K.M. and S.L.S. conducted influenza infection studies, A.P. performed LCMV infections, J.J. and C.J.T. prepared ITK inhibitors, D.L.G. provided reagents for T1D experiments, M.J.S. collaborated on fluorescence microscopy, N.J. and J.K. wrote the manuscript and J.K. and L.J.B. supervised the project.

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Correspondence to Joonsoo Kang.

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Supplementary information

Supplementary Text and Figures

Supplementary Table 1 and Supplementary Figures 1–5 (PDF 12258 kb)

Ctla4−/− T cells are highly motile in blood vessels of lung slices from B7+/+ mice.

CFSE-labeled Ctla4−/− T cells (pseudolabeled green) were intravenously injected into B7+/+ mice that were previously injected with CMTMR Orange dye (pseudolabeled red). An average of 16 frames per second was captured every 5 s using Video Savant software and a QuickTime movie rendition was made at 30 frames per second. The movie shows movement of Ctla4−/− T cells along blood vessel walls with characteristically elongated migratory morphology along vessel walls. One Ctla4−/− T cell is also shown crossing the endothelial barrier. (MOV 2974 kb)

Ctla4−/− T cells lose characteristic morphology and motility in blood vessels of lung slices from B7-deficient mice.

CFSE-labeled Ctla4−/− T cells (pseudolabeled green) were intravenously injected into Cd80−/−Cd86−/− mice that were previously injected with CMTMR Orange dye (pseudolabeled red). An average of 16 frames per second was captured every 5 s using Video Savant software and a QuickTime movie rendition was made at 30 frames per second. The movie shows the altered morphology and migratory behavior of Ctla4−/− T cells in vessel of B7-deficient mice. Ctla4−/− T cells become rounded and display random motility in the absence of B7, as opposed to directional movement in B7-sufficient lung slices. (MOV 1298 kb)

Ctla4−/− T cells are highly motile in blood vessels of lung slices from Rag1−/− mice.

CFSE-labeled Ctla4−/− T cells (pseudolabeled green) were intravenously injected into Rag1−/− mice that were previously injected with CMTMR Orange dye (pseudolabeled red). An average of 16 frames per second was captured every 5 s using Video Savant software and a QuickTime movie rendition was made at 30 frames per second. The movie shows movement of Ctla4−/− T cells with characteristically elongated migratory morphology along vessel walls; some T cells are shown moving across the endothelial cell layer into tissues while one Ctla4−/− T cell is stationery at the vessel wall. The behavior of Ctla4−/− T cells in RAG-1–deficient mice is similar to that seen in B7-sufficient RAG-1–sufficient mice (Supplementary Video 1). (MOV 2185 kb)

DKO T cells do not show migratory behavior in blood vessels of lung slices.

CFSE-labeled DKO T cells (pseudolabeled green) were intravenously injected into Rag−/− mice that were previously injected with CMTMR Orange dye (pseudolabeled red). An average of 16 frames per second was captured every 5 s using Video Savant software and a QuickTime movie rendition was made at 30 frames per second. The movie shows the behavior of DKO T cells within blood vessel of lung slices; unlike Ctla4−/− T cells, DKO T cells are rounded and do not make stable contacts with vessel walls. They do not display directionality in movement, have random motility and do not get across the endothelial barrier. (MOV 4435 kb)

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Jain, N., Miu, B., Jiang, Jk. et al. CD28 and ITK signals regulate autoreactive T cell trafficking. Nat Med 19, 1632–1637 (2013). https://doi.org/10.1038/nm.3393

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