Fig 2: Characteristics of the PD-1KD BCMA CAR-T cells. (A) Diagrams of the anti-BCMA CAR with and without shPD-1. (B) Expression of PD-1 in the CAR+cell population (9 days after virus infection) after 24 hours of coculture with 8226 cells. During coculture, shPD-1 significantly decreased the expression of PD-1 (p=0.0394 < 0.05). (C) Representative expression of PD-1 in the CAR+cell population (9 days after virus infection) determined by flow cytometry after 24 hours of coculture with 8226 cells. (D) Median fluorescence intensity (MFI) of PD-1 expression in CAR+cells detected after 24 hours of coculture with 8226 cells. CD3+ (untransfected T cells, control) or CD3+CAR+ cells were analyzed. Compared with that in BCMA CAR-T cells, shPD-1 significantly decreased the expression of PD-1 (p=0.01< 0.05). (E) Expansion of CAR-T cells after transduction (n=3 independent healthy donors). (F) Representative CAR expression was determined by flow cytometry in PD-1KD BCMA CAR-T cells (left) and BCMA CAR-T cells (right). (G) The percentages of CAR+ cells were compared between PD-1KD BCMA CAR-T cells and BCMA CAR-T cells. (H) Representative CD4+ and CD8+ CAR+T cell proportions were determined by flow cytometry in PD-1KD BCMA CAR-T cells (left) and BCMA CAR-T cells (right). (I) The ratios of CD4/CD8 in CAR+T cells were compared between PD-1KD BCMA CAR-T cells and BCMA CAR-T cells. (J) The differentiation status of PD-1KD BCMA CAR-T cells (left) and BCMA CAR-T cells (right) were determined via flow cytometry using the surface markers CD45RA and CD62L. (K) Ratios of naive (Tnaive-like; CD45RA+CD62L+), central memory (Tcm; CD45RA−CD62L+), effector memory (Tem; CD45RA−CD62L−), and most differentiated T (Temra; CD45RA+CD62 L−) cells in PD-1KD BCMA CAR-T cells and BCMA CAR-T cells were compared. BCMA, B cell maturation antigen; NS, not significant; UT, untransfected T cells.

Fig 3: Schematic outline of the suppression of NGF-promoted sympathetic axon growth by TWE-PRIL reverse signalling. (A) Model illustrating suppression of NGF-promoted axon growth by BCMA-activated TWE-PRIL reverse signalling in developing SCG neurons (represented by the semi-circle). Arrows indicate the ERK-mediated intracellular signalling that links TWE-PRIL reverse signalling to axon growth. (B-D) Summary of the main experimental manipulations that underlie this model. (B) Selective knock down of TWE-PRIL by siRNA or the absence of TWE-PRIL in April−/− mice removes the suppression signalling, resulting in enhanced NGF-promoted axon growth. (C) Knock down of BCMA by siRNA removes the activator of TWE-PRIL reverse signalling, resulting in removal of suppression signalling and enhanced NGF-promoted axon growth. (D) Soluble APRIL competes with endogenous TWE-PRIL for binding to BCMA, resulting in reduced or absent activation of TWE-PRIL reverse signalling, removal of suppression signalling and enhanced NGF-promoted axon growth.

Fig 4: Developing SCG neurons express APRIL/TWE-PRIL, TWEAK and BCMA. (A) Graphs of the levels of Twe-pril, April, Tweak, Bcma and Taci mRNAs in the SCG of E13, E15, E18, P0 and P3 mice relative to the geometric mean of reference mRNAs for glyceraldehyde phosphate dehydrogenase and succinate dehydrogenase. Data are expressed relative to the levels in adult spleen. (B) Images of dissociated P0 SCG cultures incubated for 16 h in medium containing 0.1 ng/ml NGF and 25 µM Boc-D-FMK, and triple labelled with anti-βIII tubulin antibody, the nuclear marker DAPI and antibodies that bind either APRIL/TWE-PRIL, BCMA, TWEAK or TACI. Cultures of wild-type SCG neurons double-labelled with anti-APRIL/TWE-PRIL and anti-BCMA, and the no anti-APRIL/TWE-PRIL staining controls of cultures established from April−/− mice are shown below these panels. Scale bar: 20 µm.

Fig 5: Overall analysis of BAFF system expression in LN. BAFF system expression according to glomerular, tubular, and interstitial zones is illustrated in (a–c). Black circles, black arrows, and white arrows indicate glomerular, tubular, and interstitial BAFF system expression, respectively. The overall expression analysis indicated a higher expression of BAFF and TACI than APRIL. Additionally, BAFF displays a higher expression of BR3 and BCMA (d). The p-value was obtained through Kruskal–Wallis and post hoc tests. Data are shown as the median with IQR. BAFF: B-cells activating factor, APRIL: A proliferation-inducing ligand, BR3: BAFF receptor, TACI: Transmembrane activator and CALM interactor, BCMA: B-cell maturation antigen, I IQR: Interquartile range.