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231 Dysfunctional immune synapses restrain anti-DIPG activity of CAR T cells
  1. Jorge Ibanez,
  2. Haley Houke,
  3. Michaela Meehl,
  4. Jennifer Ocasio,
  5. Nikhil Hebbar,
  6. Paulina Velasquez,
  7. Suzanne Baker and
  8. Giedre Krenciute
  1. St. Jude Children’s Research Hospital, Memphis, TN, USA


Background Diffuse intrinsic pontine gliomas (DIPGs) are highly lethal pediatric brain tumors. Thus, there is an urgent need for novel therapeutics. While chimeric antigen receptor (CAR) T-cell therapy has the potential to meet this need, early phase clinical studies with CAR T cells has shown limited antitumor activity for brain tumors. T cells require three signals for optimal activity, being (1) T cell activation, (2) costimulation, and (3) cytokines. While numerous investigators have focused on improving signals 2 and 3, the goal of this study was to investigate the role of immune synapse (IS) formation, which is critical for proper T cell activation, in the context of DIPG-targeted CAR T cell therapy.

Methods We generated GRP78-CAR T cells by expressing a 2nd generation CAR with a CD28.z signaling domain, which recognize an endoplasmic reticulum chaperone protein that is broadly expressed on the cell surface of DIPGs. We compared the effector function of GRP78-CAR T cells against U87 glioma and the patient-derived DIPG007 cell line, which are both positive for GRP78. Then we evaluated in vitro CAR T cell effector functions by MTS-based assays (cytotoxicity), repeated stimulation assays (persistence), and Milliplex (cytokine secretion). To gain mechanistic insight into tumor and CAR T cell interaction, we analyzed IS formation by live cell imaging confocal microscopy.

Results Our in vivo studies demonstrated that GRP78-CAR T cells eradicate orthotopic U87 brain tumors but have no antitumor activity against DIPG007, despite that GRP78-CAR T cells efficiently killed U87 and DIPG007 cells in vitro. However, the total cytokine production was significantly lower post DIPG007 activation (28 fold) when compared to U87 activation with IFNg, GM-CSF, TNFa, IL-2 and IL-13 being the most significantly suppressed in DIPG setting. In concordance, CAR T cells were only able to expand and retain their cytolytic activity in the presence of U87 cells. When we look at the CART:DIPG007 IS resulted in a significantly lower calcium flux in comparison to CART:U87 synapses. Likewise, the recruitment of lysosomes to CART:DIPG007 IS was significantly diminished. Importantly, we observe the same dysfunctional IS formation regardless of CAR T cell specificity and targeted antigen expression level indicating that the suppressive effect is DIPG-tumor mediated.

Conclusions Our study demonstrates that DIPG tumors suppress CAR T cell effector function by damping T cell activation through dysfunctional immune synapse formation. We are now testing other CARs and genetic engineering approaches directed at improving IS formation to overcome the suppressive effects of DIPGs.

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