Search

CD19 chimeric antigen receptor T (CD19CAR-T) cells have achieved promising outcomes in relapsed/refractory B cell malignancies. However, recurrences occur due to the loss of CAR-T cell persistence. We developed dual T/B cell co-stimulatory molecules (CD28 and CD40) in CAR-T cells to enhance intense tumoricidal activity and persistence. CD19.28.40z CAR-T cells promoted pNF-κB and pRelB downstream signaling while diminishing NFAT signaling upon antigen exposure. CD19.28.40z CAR-T cells demonstrated greater proliferation, which translated into effective anti-tumor cytotoxicity in long-term co-culture assay. Repetitive weekly antigen stimulation unveiled continuous CAR-T cell expansion while preserving central memory T cell subset and lower expression of exhaustion phenotypes. The intrinsic genes underlying CD19.28.40z CAR-T cell responses were compared with conventional CARs and demonstrated the up-regulated genes associated with T cell proliferation and memory as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway. Enrichment of genes toward T cell stemness, particularly SELL, IL-7r, TCF7, and KLF2, was observed. Effective and continuing anti-tumor cytotoxicity in vivo was exhibited in both B cell lymphoblastic leukemia and B cell non-Hodgkin lymphoma xenograft models while demonstrating persistent T cell memory signatures. The functional enhancement of CD37.28.40z CAR-T cell activities against CD37+ tumor cells was further validated. The modification of dual T/B cell signaling molecules remarkably maximized the efficacy of CAR-T cell therapy.

Summary: T cell co-inhibitory immune checkpoints, such as PD-1 or BTLA, are bona fide targets in cancer therapy. We used a human T cell reporter line to measure transcriptomic changes mediated by PD-1- and BTLA-induced signaling. T cell receptor (TCR)-complex stimulation resulted in the upregulation of a large number of genes but also in repression of a similar number of genes. PD-1 and BTLA signals attenuated transcriptomic changes mediated by TCR-complex signaling: upregulated genes tended to be suppressed and the expression of a significant number of downregulated genes was higher during PD-1 or BTLA signaling. BTLA was a significantly stronger attenuator of TCR-complex-induced transcriptome changes than PD-1. A strong overlap between genes that were regulated indicated quantitative rather than qualitative differences between these receptors. In line with their function as attenuators of TCR-complex-mediated changes, we found strongly regulated genes to be prime targets of PD-1 and BTLA signaling.

Summary: Costimulatory signals provided to T cells during antigen encounter have a decisive role in the outcome of immune responses. Here, we used chimeric receptors harboring the extracellular domain of mouse inducible T cell costimulator (mICOS) to study transcriptional activation mediated by cytoplasmic sequences of the major T cell costimulatory receptors CD28, 4-1BB, and CD2. The chimeric receptors were introduced in a T cell reporter platform that allows to simultaneously evaluate nuclear factor κB (NF-κB), NFAT, and AP-1 activation. Engagement of the chimeric receptors induced distinct transcriptional profiles. CD28 signaling activated all three transcription factors, whereas 4-1BB strongly promoted NF-κB and AP-1 but downregulated NFAT activity. CD2 signals resulted in the strongest upregulation of NFAT. Transcriptome analysis revealed pronounced and distinct gene expression signatures upon CD2 and 4-1BB signaling. Using the intracellular sequence of CD28, we exemplify that distinct signaling motifs endow chimeric receptors with different costimulatory capacities.

The receptor for advanced glycation end products (RAGE) is encoded by AGER, a gene that is subjected to tissue-specific alternative splicing. Splice variants of RAGE in intestine and placenta are unknown and contradictory data concerning RAGE protein expression in these tissues have been published. As a basis for future functional studies, we examined RAGE expression in small intestine, colon and placentas. PCR cloning revealed that full-length RAGE is the only RAGE transcript isoform expressed in placenta. In the small intestine, the major transcript isoform detected was RAGE_v1 encoding the C-terminally truncated soluble receptor. In the colon, both full-length RAGE as well as several splice variants were identified. Four antibodies were used to study protein expression by immunoblotting and were carefully validated. Appropriate controls were essential to avoid misinterpretation of bands caused by non-specific reactivity of antibodies. Only one of four antibodies tested detected full-length RAGE in placenta, whereas no RAGE-specific band was detected in intestinal tissues despite loading >30-fold more intestinal tissue than the positive control, human lung. RAGE expression levels in the placenta were 100-fold lower compared with human lung when analyzed by ELISA, and no significant differences in RAGE expression were detected between healthy placentas and placentas from women with preeclampsia, gestational diabetes mellitus, or fetal growth restriction. We conclude that healthy placental chorionic tissue expresses low levels of full-length RAGE, whereas expression of the tissue-specific intestinal isoforms is below the limit of detection. Low RAGE expression levels in combination with a lack of antibody validation may explain the conflicting published results on RAGE protein expression in intestine and placenta.

An artificial T cell adaptor molecule (ATAM) was generated to improve persistence of T cell receptor (TCR) gene-transduced T (TCR-T) cells compared to such persistence in a preceding study. ATAMs are gene-modified CD3ζ with the intracellular domain of 4-1BB inserted in the middle of CD3ζ. NY-ESO-1 TCR-T cells transduced with an ATAM with two separated virus vectors demonstrated superior proliferation upon antigen stimulation. To further develop clinically applicable ATAM-transduced TCR-T cells, we attempted to make a single virus vector to transduce the TCR and ATAM simultaneously. Because we failed to observe improved proliferation capacity upon stimulation after one virus vector (1vv) transduction, we compared TCR-T cells transduced with 1vv and two virus vector (2vv) methods to elucidate the reason. In Jurkat reporter cells, an ATAM transduced by the 2vv method demonstrated a higher intensity than by the 1vv method, and the ATAM intensity was associated with increased nuclear factor κB (NF-κB) signals upon stimulation. In ATAM-transduced primary T cells, a transduced ATAM by the 2vv method showed higher intensity and better proliferation. ATAM-transduced TCR-T cells demonstrated improved proliferation only when the ATAM was transduced at a higher intensity. To create a simpler transduction method, we need to develop a strategy to make a higher ATAM expression to prove the efficacy of ATAM transduction in TCR-T therapy.