Katie M. Campbell

T-cell exhaustion limits durable antitumor responses and remains a barrier to adoptive cell therapies (ACT) in solid tumors. To identify regulators of T-cell persistence, we performed genome-wide CRISPR knockout screens in primary human CD8+ T cells expressing an NY-ESO-1–specific T-cell receptor and subjected them to repeat tumor cell rechallenges. These screens revealed an enrichment of single-guide RNAs targeting components of cullin–RING E3 ubiquitin ligases, particularly cullin-5–RING E3 ligase (CRL5) assemblies comprising CUL5, RNF7, and ARIH2. Disruption of CRL5 genes in T cells preserved cytotoxic activity and enhanced cytokine production while sustaining effector-associated transcriptional programs and suppressing exhaustion-related signatures. Pharmacologic inhibition of cullin neddylation with the NEDD8-activating enzyme inhibitor MLN4924 recapitulated CRL5 loss and enhanced T-cell effector function in vitro. In a human melanoma xenograft model, ACT with CRL5-deficient T cells improved tumor control. Together, these findings identify CRL5 as a negative regulator of T-cell fitness and a potential therapeutic target to enhance ACT durability in solid tumors. Significance: The CRL5 complex negatively regulates CD8+ T-cell persistence during chronic antigen stimulation, identifying CRL5 disruption as a potential strategy to sustain T-cell effector function and improve ACT. See related commentary by Hanina and Maus, p. XX . See related article by Bachl et al., p. XX .

Spatial multi-omic technologies entail simultaneous measurement of tens-to-thousands of molecular features (e.g. RNA, protein). While cell type and state profiling is the major focus of spatial multi-omic analyses, cytoskeletal proteins are often solely utilized for cell segmentation and quality control. Here, we introduce a new computational framework, CytoRipple, that leverages these concomitant spatially-resolved single-cell measurements of cytoskeleton proteins to identify tissue-level vector fields. Polarization vectors are known to associate with cell movement and phenotype, inferring their physical orientation. We validate these associations across biological contexts and reveal how polarization vector fields can be harnessed to discover multi-cellular structures (e.g. “streams” of cells with similar polarizations). These multi-cellular cytoskeleton dynamics associate with distinct phenomena, such as vector “divergence” with CD8 ⁺ T cell activation and “streams” with spatially segmented stages of the anti-tumor immune response. Together, these data support a role for CytoRipple in revealing a dynamic layer across spatial multi-omic platforms.

Outcomes for high-grade pediatric brain tumor patients remain poor, but there is optimism that chimeric antigen receptor (CAR) T cell therapy can improve prognosis. We present the results from a phase I clinical trial of IL13BBζ-CAR T cells infused weekly into the cerebral ventricles in pediatric and young adult patients with recurrent or refractory brain tumors. The trial met its primary objectives of feasibility, safety, and tolerability, with one dose-limiting toxicity. 8 of 16 patients evaluable for response experienced radiographic size decreases consistent with biologic activity and with an anti-tumor response. Two patients met protocol criteria for response. Median survival for patients receiving lymphodepletion was 20.5 months from diagnosis and 6.9 months from treatment for patients with midline glioma, and 187 months from diagnosis and 7.5 months from treatment for patients with ependymoma. Importantly, patients who did not receive lymphodepletion developed anti-CAR humoral and cellular immune responses detectable in the CSF and peripheral blood, whereas patients receiving lymphodepletion had no evidence of CSF anti-CAR immunity. Taken together, these findings demonstrate the safety, tolerability, and biological activity of locoregionally-delivered IL13BBζ-CAR T cells for children and young adults with CNS tumors. Moreover, we show that anti-CAR immune responses arise in patients not receiving lymphodepletion, but not in the CSF of patients receiving systemic lymphodepletion. Further investigation of adoptive cellular therapies combined with immunosuppression is warranted in this patient population. ClinicalTrials.gov registration: NCT04510051 .

Supplementary Figure S2 shows the genomic variants detected by WES and statistical analaysis of WES data.

Supplementary Figure S3 shows the annotation of cells evaluated by NanoString.

Supplementary Figure S1 shows the processing and review of sample processing for WES, RNAseq, and MIBI.

Supplementary Figure S4 show the comparison of cell types by MIBI across sample/response groups.

Supplementary Figure S5 shows the expression of protein markers by cell type subsets in MIBI data.

Supplementary Figure S6 shows the TCR/BCR repertoire metrics in RNAseq data from biopsies from patients nonresponsive to combination therapy.

Supplementary Figure S7 shows the evaluation of TLS signatures in RNAseq and MIBI samples.