The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor–armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. Significance CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

Activation of a naive T cell is a highly energetic event, which requires a substantial increase in nutrient metabolism. Upon stimulation, T cells increase in size, rapidly proliferate, and differentiate, all of which lead to a high demand for energetic and biosynthetic precursors. Although amino acids are the basic building blocks of protein biosynthesis and contribute to many other metabolic processes, the role of amino acid metabolism in T cell activation has not been well characterized. We have found that glutamine in particular is required for T cell function. Depletion of glutamine blocks proliferation and cytokine production, and this cannot be rescued by supplying biosynthetic precursors of glutamine. Correlating with the absolute requirement for glutamine, T cell activation induces a large increase in glutamine import, but not glutamate import, and this increase is CD28-dependent. Activation coordinately enhances expression of glutamine transporters and activities of enzymes required to allow the use of glutamine as a Krebs cycle substrate in T cells. The induction of glutamine uptake and metabolism requires ERK function, providing a link to TCR signaling. Together, these data indicate that regulation of glutamine use is an important component of T cell activation. Thus, a better understanding of glutamine sensing and use in T cells may reveal novel targets for immunomodulation.

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