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Role of CD28 in PD-1-mediated inhibition and the rescue of exhausted T cells during PD-1-targeted therapy

June 21, 2017

Although the PD-1-targeted therapies have provided clinical benefit in a variety cancers, the mechanism behind PD-1-mediated inhibition of T cells, and their subsequent rescue with PD-1-targeted therapy, is not well understood. In two separate studies, Kamphorst et al. and Hui et al. demonstrate that the CD28 costimulatory pathway plays a surprisingly important role in the PD-1 effect on T cell function and the rescue of exhausted CD8+ T cells during PD-1-targeted therapy.

Using the chronic lymphocytic choriomeningitis virus (LCMV) infection mouse model, Kamphorst et al. utilized several methods to block the CD28/B7 costimulatory pathway along with blockade of the PD-1/PD-L1 axis. When the CD28/B7 pathway was blocked, virus-specific CD8+ T cells failed to expand or restore effector function, which was in contrast to the successful rescue observed with PD-1 blockade alone. CD8+ T cells that were either intrinsically CD28-deficient or that lost CD28 during exhaustion could not be rescued with PD-1 blockade.

The same team then examined the role of the CD28/B7 pathway in the CD8+ T cell anti-tumor response in mice with colon carcinoma that had been administered anti-PD-L1 therapy. They observed tumor regression with anti-PD-L1, but tumor progression with additional blockade of the CD28/B7 axis, thus confirming the importance of CD28 costimulation in anti-tumor response.

Samples from advanced non-small cell lung cancer (NSCLC) patients undergoing anti-PD-1 treatment indicated that the PD-1+CD8+ T cells activated by therapy were mostly CD28+, suggesting that CD28 signaling is involved in the PD-1 blockade response in human cancer. Examination of tumor samples from early stage NSCLC patients uncovered that many human CD8+ tumor infiltrating lymphocytes do not express CD28, and therefore may not respond well to PD-1-targeted therapy.

While the preclinical studies by Kamphorst et al. discovered that CD28 costimulation plays a role in PD-1-targeted therapy, Hui et al. explained the mechanism behind CD28 involvement in the PD-1 pathway. This team of researchers biochemically explored the intracellular interactions of PD-1, and found that CD28, rather than the TCR, is the primary target of PD-1-mediated inhibition of T cell activation.

The team used a cell-free biochemical reconstitution system, which included large unilamellar vesicles that represented the T cell plasma membrane, with the cytoplasmic domain of PD-1 attached to their surface. Utilizing fluorescence resonance energy transfer (FRET), researchers discovered that the TCR-phosphorylating kinase Lck phosphorylated two tyrosines on PD-1 in vitro upon the addition of ATP. (In vivo, this process is triggered by the binding of PD-1 to PD-L1.) The phosphorylated PD-1 directly bound the tyrosine phosphatase Shp2 (but not any other SH2 domain-containing proteins that were tested), leading to inhibitory signaling.

Using a titration system that comprised many of the T cell signaling components, the team discovered that CD28 (but not the TCR or its associated components) was the most efficiently dephosphorylated substrate of the PD1-Shp2 complex. Similar results were observed in intact T cells, with the notable difference that the dephosphorylation of CD28 initiated by PD-1-PD-L1 binding was transient (much higher at 2 minutes than at 10 minutes after APC/T cell contact), likely due to a feedback loop involving the closely associated Lck kinase.

Together, the results of the two studies demonstrate that CD28 costimulation is required for effective PD-1-targeted therapy and suggest that CD28 may be useful as a biomarker to predict treatment response in cancer patients. As activation of CD28 signaling requires ligation with B7 molecules, these results strongly implicate a critical role for antigen-presenting cells (or another source of CD28 stimulation) in effective anti-PD-1 axis therapy, and highlight the importance of fully understanding the integration of positive and negative signaling events that form the basis for T cell activity.

by Anna Scherer


Kamphorst A.O., Wieland A., Nasti T., Yang S., Zhang R., Barber D.L., Konieczny B.T., Daugherty C.Z., Koenig L., Yu K., Sica G.L., Sharpe A.H., Freeman G.J., Blazar B.R., Turka L.A., Owonikoko T.K., Pillai R.N., Ramalingam S.S., Araki K., Ahmed R. Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent. Science. 2017 Mar 31.

Hui E., Cheung J., Zhu J., Su X., Taylor M.J., Wallweber H.A., Sasmal D.K., Huang J., Kim J.M., Mellman I., Vale R.D. T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Science. 2017 Mar 31.

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