Data collection
The mRNA profiling and clinical data (age, gender, WBC, and CNS status) were obtained from the database of Gene Expression Omnibus (GEO http://www.ncbi.nlm.nih.gov/geo, accession number: GSE11877). Cytogenetic abnormalities of 134 patients were obtained from Mullighan[10], which excluded the cases with known very high-riskALL subtypes (i.e., BCR-ABL1-positive ALL, hypodiploid ALL, ETV6-RUNX1, and ALL in infants).These patients included 70 cases with PAX5 mutations and 64cases without PAX5 mutations.
Assessment of immune cell types
As a deconvolution algorithm, CIBERSORT can estimate the cell composition of complex tissues based on normalized GEPs. We used CIBERSORT (http://cibersort.stanford.edu/) to estimate the relative fractions of immune cell types in bone marrow of B-ALL. These immune cell types in bone marrow included Tcells(Tfh cells, resting memory CD4 + T cells, activated memory CD4 + T cells, γδ T cells, CD8 + T cells, Tregs, naïve CD4 + T cells), B cells (memory B cells, naïve B cells, plasma cells), NK cells (activated and resting NK cells), and myeloid subsets (M0 macrophages, M1 macrophages, M2 macrophages, activated and resting mast cells, activated and resting dendritic cells, neutrophils, monocytes and eosinophils) [19].
Cell culture
The BALB/c B cell lymphoma line A20 was purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China) and HEK293T was purchased from CCTCC (Wuhan, China). A20 cells were cultured in RPMI1640 medium containing 5–10% fetal calf serum (FCS, Invitrogen, United States), HEK293T cells were cultured in DMEM supplemented with 10% dialyzed fetal bovine serum.
Construction and identification of PAX5+/-A20 cell clone
Construction and identification of PAX5+/-A20 cell clone were carried out in a way that we previously established [44].
The vector pSpCas9(BB) -2A-GFP (PX458)(Plasmid #48138)was bought from Addgene (Massachusetts, United States). The oligo DNA targeting the PAX5 exon3 locus was designed on the MIT online software ZhangFeng lab: http://crispr.mit.edu/. The primers used for knockout were synthesized in Invitrogen company (see the Supplementary Appendix Table 1). We formed dsDNA using Precut sgRNA Cloning kit and pSDgRNA Plasmid construction Kit (Biomics Biotechnologies Co., Ltd, Jiangsu, China) according to the instruction. Next, Cas9/sgRNA plasmids targeting PAX5 were constructed, amplified, purified with EndoFree Plasmid Maxi Kit (QIAGEN, Germany), and were validated by sequencing. The Cas9/sgRNA plasmids were electrotransfected into HEK293T cells. The T7 Endonuclease I assay was applied to survey the efficiency of NHEJ-mediated mutations in the endogenous PAX5 gene. The most efficient Cas9/sgRNA plasmid was chosen for follow-up research.
Table 1
Clinical features of the patient cohorts
Clinical Features | PAX5+(n = 70) | PAX5- (n = 64) | p value |
Gender | | | ༞0.05 |
Male | 46(66%) | 43(67%) | |
Female | 24(34%) | 21(33%) | |
Age (months) | | | ༞0.05 |
Median | 146 | 171 | |
Range | 17–249 | 12–222 | |
WBC (1000/ul) | 105 ± 118 | 103 ± 145 | ༞0.05 |
CNS disease | 16(23%) | 12(19%) | ༞0.05 |
PAX5+: with PAX5 alteration, PAX5-: without PAX5 alteration |
3 × 105 A20 cells were collected and suspended with the matched solution supplemented with 5 µg CRISPR/CAS9 plasmid. Electrotransfection was performed on LONZA 4D Nucleofector System (Lonza, Switzerland). Cells were cultured for 48 h and then sorted with Beckman MoFlo XDP (Beckman Coulter, Inc., American), aiming to select cells with high GFPs expression. The sorted cells were seeded in 96-well plates in the manner of a single cell. 2–3 weeks’ later, cells were collected for identification. Genomic DNA was isolated using the DNA Isolation Kit (BioTeke Corporation, Beijing, China), and PAX5 gene was amplified with PCR for mutation analysis. The PCR primers were synthesized by Sangon Biotech (Shanghai, China) (see the Supplementary Appendix Table 2). The PCR product was reclaimed for sequence determination. The sequencing results were compared with the published PAX5 gene sequence to determine the presence of PAX5+/- variants.
Cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) added with a protease inhibitor cocktail (Roche, Switzerland). The protein concentration was measured by the cinchoninic acid protein assay (Thermo Scientific, United States). 30 µg of total lysate was subjected to SDS-PAGE and then transferred to nitrocellulose membranes (Bio-Rad Laboratories, United States). The membranes were incubated with the antibodies against PAX5 and ß-actin purchased from Abcom Biotechnology (Abcam, CA, United States), and they were blotted with corresponding HRPlinked secondary antibodies. The proteins were measured using an enhanced ECL system (Pierce, United States). Quantification was performed with ImageJ (https://imagej.nih.gov/) and each sample's ratio relative to the loading control β-actin was calculated.
Structuring allografted tumor models
All methods were fulfilled based on the Guidelines for the Care and Use of Laboratory Animals of the Council of Science and Technology of China. All test protocols were approved by the Animal Care and Use Committee of Tongji Hospital. Six-weeks old female BALB/c mice were bought from the Animal Center of Tongji Medical College. All mice were fed in individually ventilated, pathogen-free cages where they had access to food and water optionally. Tumor size, weight, activity, and skin changes were monitored carefully throughout the experiments. No animals showed signs of illness following tumor formation and none died due to the experimental procedure.
Forty mice were divided at random into four groups: PAX5+/- mice (MUT1, n = 10; MUT2, n = 10; MUT3, n = 10) and PAX5 wild type mice (WT,n = 10). 5 × 106 cells in 0.15 ml PBS were subcutaneously injected into the belly of BALB/c mice. The growth of the allografted tumors was observed for two weeks. After two weeks, the mice were fully anesthetized using Ketalar-Dormicum and sacrificed with CO2. We observed whether allografted tumors formed and tumor weight was measured. And then, Tumor specimens were used for the next research.
Sorting of CD8 + T cells
Tumors were cut into small fragments and digested, and single-cell suspensions were prepared. CD8 + T cells were sorted using anti-mouse CD8a MicroBeads (Miltenyi Biotec Inc, United States) according to the manufacturer’s instructions. The purity of CD8 + T cells was typically greater than 90% by fluorescence activating cell sorter.
Quantitative RT-PCR
Total RNA was extracted with TRIzol reagent (Invitrogen, United States), and was reverse transcribed into cDNA using the Hieff cDNA Synthesis Super Mix (Yeasen Biotech, Shanghai, China). All primers were shown (see the Supplementary Appendix Table 2). The Applied Bio-systems StepOneTM RT-PCR machine was used to carry out quantitative PCR with the Hieff qPCR SYBR Green Master Mix (Yeasen Biotech, Shanghai, China). All reactions were repeated at least in triplicate. We used endogenous GAPDH to normalize the sample with the comparative threshold cycle (Ct) method for relative quantification.
Flow cytometry
To quantify levels of immune inhibitory molecules of CD8 + T cells in tumor specimens, single cell suspensions were surface or intracellular stained with the following fluorescent antibodies:CD45-APC-cy7, CD3-APC, CD8-PerCP, TIM3-PE-Cy7, BATF-PE, NR4A1-FITC, IFN-ɤ-BV510(BD Biosciences,Germany). For BATF,NR4A1 staining, cells were fixed and permeabilized with the Transcription Factor staining buffer kit(Invitrogen, United States)according to the manufacturer’s instructions. For IFN-ɤ staining, single-cell suspensions were first stimulated with cell stimulation cocktail(500×༉(Invitrogen, United States༉and inhibited with protein transport inhibitor(500×༉(Invitrogen, United States), and then fixed and permeabilized with Cytofix/Cytoperm Fixation/Permeabilization Solution Kit(BD Biosciences,Germany).Stained samples were collected and analyzed using a FACScan flow cytometer (BD Biosciences,Germany). Finally, all the data were analyzed by the FlowJo software (v10,United States).
Statistical analysis
Statistical analyses of the mRNA profiling from B-ALL were conducted using the “CIBERSORT.R” package and Bioconductor (https://www.bioconductor.org/). Each dataset was processed by a weighted average method to compare the differences in the composition of immune cell types and the mRNA levels of immune inhibitory -related molecules between cases with PAX5 mutations and cases without PAX5 mutations. All analyses were conducted using R Studio (version 4.0.3).
The results from mice were expressed as the mean and standard deviation (SD) of at least three independent experiments and three repetitions. Statistical significance was estimated by Student's t-tests or one-way analysis of variance (ANOVA) followed by Dunnett's test. The statistical analyses were performed using the Graphpad Prism (version 8.0) software. A p-value of < 0.05 was significant.