Androgen receptor activity inversely correlates with immune cell infiltration and immunotherapy response across multiple cancer lineages.

There is now increasing recognition of the important role of androgen receptor (AR) in modulating immune function. To gain a comprehensive understanding of the effects of AR activity on cancer immunity, we employed a computational approach to profile AR activity in 33 human tumor types using RNA-Seq datasets from The Cancer Genome Atlas. Our pan-cancer analysis revealed that the genes most negatively correlated with AR activity across cancers are involved in active immune system processes. Importantly, we observed a significant negative correlation between AR activity and IFNγ pathway activity at the pan-cancer level. Indeed, using a matched biopsy dataset from subjects with prostate cancer before and after AR-targeted treatment, we verified that inhibiting AR enriches immune cell abundances and is associated with higher IFNγ pathway activity. Furthermore, by analyzing immunotherapy datasets in multiple cancers, our results demonstrate that low AR activity was significantly associated with a favorable response to immunotherapy. Together, our data provide a comprehensive assessment of the relationship between AR signaling and tumor immunity.

Androgen blockade primes NLRP3 in macrophages to induce tumor phagocytosis.

Immune-based therapies induce durable remissions in subsets of patients across multiple malignancies. However, there is limited efficacy of immunotherapy in metastatic castrate-resistant prostate cancer (mCRPC), manifested by an enrichment of immunosuppressive (M2) tumor- associated macrophages (TAM) in the tumor immune microenvironment (TME). Therefore, therapeutic strategies to overcome TAM-mediated immunosuppression are critically needed in mCRPC. Here we discovered that NLR family pyrin domain containing 3 (NLRP3), an innate immune sensing protein, is highly expressed in TAM from metastatic PC patients treated with standard-of-care androgen deprivation therapy (ADT). Importantly, ex vivo studies revealed that androgen receptor (AR) blockade in TAM upregulates NLRP3 expression, but not inflammasome activity, and concurrent AR blockade/NLRP3 agonist (NLRP3a) treatment promotes cancer cell phagocytosis by immunosuppressive M2 TAM. In contrast, NLRP3a monotherapy was sufficient to enhance phagocytosis of cancer cells in anti-tumor (M1) TAM, which exhibit high de novo NLRP3 expression. Critically, combinatorial treatment with ADT/NLRP3a in a murine model of advanced PC resulted in significant tumor control, with tumor clearance in 55% of mice via TAM phagocytosis. Collectively, our results demonstrate NLRP3 as an AR-regulated "macrophage phagocytic checkpoint", inducibly expressed in TAM by ADT and activated by NLRP3a treatment, the combination resulting in TAM-mediated phagocytosis and tumor control.

LSD1 promotes prostate cancer reprogramming by repressing TP53 signaling independently of its demethylase function.

Lysine-specific demethylase 1 (LSD1) is a histone demethylase that promotes stemness and cell survival in cancers such as prostate cancer. Most prostate malignancies are adenocarcinomas with luminal differentiation. However, some tumors undergo cellular reprogramming to a more lethal subset termed neuroendocrine prostate cancer (NEPC) with neuronal differentiation. The frequency of NEPC is increasing since the widespread use of potent androgen receptor signaling inhibitors. Currently, there are no effective treatments for NEPC. We previously determined that LSD1 promotes survival of prostate adenocarcinoma tumors. However, the role of LSD1 in NEPC is unknown. Here, we determined that LSD1 is highly upregulated in NEPC versus adenocarcinoma patient tumors. LSD1 suppression with RNAi or allosteric LSD1 inhibitors - but not catalytic inhibitors - reduced NEPC cell survival. RNA-Seq analysis revealed that LSD1 represses pathways linked to luminal differentiation, and TP53 was the top reactivated pathway. We confirmed that LSD1 suppressed the TP53 pathway by reducing TP53 occupancy at target genes while LSD1's catalytic function was dispensable for this effect. Mechanistically, LSD1 inhibition disrupted LSD1-HDAC interactions, increasing histone acetylation at TP53 targets. Finally, LSD1 inhibition suppressed NEPC tumor growth in vivo. These findings suggest that blocking LSD1's noncatalytic function may be a promising treatment strategy for NEPC.

Transcriptional profiling of matched patient biopsies clarifies molecular determinants of enzalutamide-induced lineage plasticity.

The androgen receptor (AR) signaling inhibitor enzalutamide (enza) is one of the principal treatments for metastatic castration-resistant prostate cancer (CRPC). Several emergent enza clinical resistance mechanisms have been described, including lineage plasticity in which the tumors manifest reduced dependency on the AR. To improve our understanding of enza resistance, herein we analyze the transcriptomes of matched biopsies from men with metastatic CRPC obtained prior to treatment and at progression (n = 21). RNA-sequencing analysis demonstrates that enza does not induce marked, sustained changes in the tumor transcriptome in most patients. However, three patients' progression biopsies show evidence of lineage plasticity. The transcription factor E2F1 and pathways linked to tumor stemness are highly activated in baseline biopsies from patients whose tumors undergo lineage plasticity. We find a gene signature enriched in these baseline biopsies that is strongly associated with poor survival in independent patient cohorts and with risk of castration-induced lineage plasticity in patient-derived xenograft models, suggesting that tumors harboring this gene expression program may be at particular risk for resistance mediated by lineage plasticity and poor outcomes.

Glutamine Administration in Early or Late Septic Phase Downregulates Lymphocyte PD-1/PD-L1 Expression and the Inflammatory Response in Mice With Polymicrobial Sepsis.

BACKGROUND: Sepsis is a severe inflammatory response to infection. Excessive compensation to inflammation leads to dysregulated immune response that ultimately results in organ damage and lethality of sepsis. This study administered glutamine (GLN) in the early or late phase of sepsis to investigate its effects on regulating leukocyte programmed cell death 1 (PD-1) and its ligand (programmed cell death ligand 1 [PD-L1]) expression, macrophage function, inflammation, and acute kidney injury in sepsis. METHODS: Mice were randomly assigned to cecal ligation and puncture (CLP) or sham-operated groups. Septic mice were respectively injected once with saline or 0.75 g GLN/kg body weight at 3 or 10 hours post-CLP via tail vein. All mice were sacrificed 24 hours after CLP. RESULTS: Sepsis enhanced the percentage of interferon-γ-expressing and interleukin (IL)-17A-expressing CD4+ T cells, expression of PD-1 on T cells, and PD-L1 on B cells and monocytes. Inflammatory mediator messenger RNA (mRNA) expression in kidney tissues and proapoptotic caspase-3 mRNA expression in mesenteric lymph nodes were also upregulated. GLN administration decreased plasma IL-6 level, downregulated the percentage of IL-17A-expressing CD4+ T cells, attenuated macrophage dysfunction, decreased caspase-3 mRNA expression, and reduced PD-1/PD-L1 expression by T and B cells. Histological findings also showed that kidney damage was attenuated. GLN administered at 3 and 10 hours after CLP offered nearly equal effects on PD-1/PD-L1 and inflammatory mediator expression after CLP. CONCLUSIONS: These findings suggest that a single dose of GLN administration in either the early or late phase during sepsis promotes a more balanced immune regulation and reduced systemic and kidney inflammatory responses in mice.

Glutamine modulates sepsis-induced changes to intestinal intraepithelial γδT lymphocyte expression in mice.

This study investigated the effect of glutamine (GLN) on intestinal intraepithelial lymphocyte (IEL) γδT-cell cytokines and immune regulatory factor gene expressions in a mouse model of polymicrobial sepsis. Mice were randomly assigned to a normal group, a sepsis with saline (SS) group, or a sepsis with GLN (SG) group. All mice were fed a chow diet. Sepsis was induced by cecal ligation and puncture (CLP). The SS group was injected with saline, and the SG group was given 0.75 g GLN/kg body weight once via a tail vein 1 h after CLP. Septic mice were killed 12 h after CLP, and IEL γδT cells of the animals were isolated for further analysis. Results showed that compared with normal mice, sepsis resulted in lower IEL γδT-cell percentage and higher messenger RNA expressions of interferon γ, tumor necrosis factor α, interleukin 4 (IL-4), IL-13, IL-17, retinoid acid receptor-related orphan receptor γt, and complement 5a receptor by IEL γδT cells. These immunomodulatory mediator genes exhibited decreases, whereas IL-7 receptor expression increased in IEL γδT cells in septic mice with GLN administration. Annexin V/7-amino-actinomycin D stain revealed significantly lower rates of apoptosis, and IEL γδT-cell percentage was higher in the SG group. The histological findings also showed that damage to intestinal epithelial cells was less severe in the SG group. These results indicated that a single dose of GLN administered as treatment after the initiation of sepsis prevented apoptosis of IEL γδT cells and downregulated γδT cell-expressed inflammatory mediators that may consequently ameliorate the severity of sepsis-induced intestinal epithelial injury.

Effects of supplemental dietary arginine on the exogenous advanced glycosylation end product-induced interleukin-23/interleukin-17 immune response in rats.

OBJECTIVES: Arginine (Arg) is known to possess numerous useful physiological properties and immunomodulatory effects. Th17 cells are a unique T-helper cell lineage. Regulation of Th17 cells plays a significant role in the pathogenesis of inflammatory disorders. This study investigated the effect of Arg on the exogenous advanced glycosylation end product (AGE)-induced Th17-mediated immune response. METHODS: Rats were randomly divided into three groups. The control BSA (CB) group was fed a common diet and given a tail vein injection of non-glycated bovine serum albumin (BSA). The control AGE (CA) group was fed the common diet and injected with 2 mg AGE-BSA. Arg-AGE (AA) group was fed the Arg-supplemented diet and injected with 2 mg AGE-BSA. The experimental diets were identical in energy and nutrient distributions except for the amino acid content. Arg provided 2% of the total energy. Tail vein injections and diets were given daily. After 10 d, all rats were sacrificed, and blood samples were collected for further analysis. RESULTS: The AA group had the highest inducible nitric oxide (NO) synthase expression and plasma NO levels. The percentage of Foxp3 T-regulatory cells in the AA group was lower than those of the CA and CB groups. Transforming growth factor-ß1, interleukin (IL)-6, and IL-17A gene expression was higher in the AGE-administered groups. The AA group had higher TGF-ß1 and IL-17A expression than did the CA group. CONCLUSION: These results suggest that in a condition of exogenous AGE administration, supplemental dietary Arg resulted in a more pronounced IL-23/IL-17 immune response, possibly by increasing NO secretion.

[Cloning and expression of pokeweed antiviral protein gene from Phytolacca americana in Pichia pastoris and the study of apoptosis of human neuroglioma cells U251 induced by recombinant PAP].

OBJECTIVE: To clone the pokeweed anti-viral protein (PAP) gene, to express it in Pichia pastoris, and to study the inhibitory effect of PAP on U251 in vitro. METHODS: The cDNA sequence encoding PAP was cloned by Real-time PCR from Phytolacca americana. The recombinant PAP was subcloned into the expression vector pPICZaA and expressed in Pichia pastoris GSI15 after methanol induction. SDS-PAGE analysis showed that the expressed PAP existed in the yeast culture supernatant. The drug cytotoxicity to U251 cells was assessed using MTT assay and the obvious apoptotic nuclei of the tumor cells detected using the method of single cell gel electrophoresis. RESULTS: The full-length PAP gene was cloned. The recombinant expression plasmid pPICZaA-PAP was constructed successfully. SDS-PAGE analysis showed that the relative molecular mass (M) of the recombinant protein was about 35 kDa. The degradation of the genome of the apoptotic cells induced by PAP was detected using the method of single cell gel electrophoresis. PAP possessed very high ability to inhibit the growth of U251. The anti-tumor activities (IC50) to U251 cells of PAP was 81.0 microg/mL. CONCLUSION: PAP could be a potent anti-tumor candidate for inhibiting the growth of U251 and inducing its apoptosis.

Effects of dietary glutamine supplementation on lung injury induced by lipopolysaccharide administration.

Acute lung injury (ALI) is a critical syndrome associated with respiratory dysfunction, and neutrophils are considered to be central to the pathogenesis of ALI. This study investigated the effects of glutamine (Gln) on neutrophil recruitment in a model of lipopolysaccharide (LPS)-induced ALI. C57BL/6 mice were fed a standard diet either with casein as the nitrogen source or with 25% of total nitrogen replaced by Gln. After 10 days, intratracheal instillation of LPS was used to induce ALI. Mice were killed at 0, 6, 12, and 24 h after LPS administration (n = 10/group). Bronchoalveolar lavage fluid and lung tissues were collected for further analysis. The results showed that, compared with the control group, lipid peroxide levels in the lungs were higher at 12 and 24 h after LPS administration in the Gln group. CXC chemokines as well as tumor necrosis factor-alpha were significantly elevated and reached peaks at 6 h in the Gln group, which was earlier than in the control group. Histopathological findings showed that the thickening of alveolar septal space was extensive in the Gln group 24 h and 2 wk after LPS. Also, greater amounts of collagen had accumulated in lung tissue in the Gln group. This study indicates that dietary Gln administration resulted in higher inflammatory cytokine production, with more neutrophils recruited at the early stage of ALI. These results were consistent with the histopathological findings that Gln supplementation causes more severe interstitial inflammation and fibrosis in a model of ALI induced by LPS.

Effects of dietary fish oil supplementation on cellular adhesion molecule expression and tissue myeloperoxidase activity in hypercholesterolemic mice with sepsis.

This study investigated the effects of fish oil on adhesion molecule expression and tissue myeloperoxidase (MPO) activity in hypercholesterolemic mice with sepsis. There were one control and two experimental groups in this study. The control group (C) was fed a regular chow diet for 7 weeks, while hypercholesterolemia in the experimental group was induced by feeding a high-fat diet (20%, w/w) with cholesterol (2%, w/w) for 4 weeks. Then the experimental group was divided into two subgroups with identical nutrient distributions except that one subgroup was fed soybean oil (SO), while part of the soybean oil was replaced by fish oil (FO) in the other one for 3 weeks. After feeding the diets for 7 weeks, sepsis was induced in all three groups by cecal and ligation and puncture (CLP), and mice were sacrificed at 0, 6 or 24 h after CLP, respectively. The results showed that the FO group had a higher intracellular interferon-gamma/interleukin-4 ratio and lower tumor necrosis factor-alpha and monocyte chemoattractant protein-1 concentrations in peritoneal lavage fluid at 6 h after CLP than those in the C and SO groups. Lymphocyte CD11a/CD18 expressions were higher at 0 and 6 h and neutrophil CD11b/CD18 were higher at 6 h in the SO group than in the FO and C groups. The SO group had higher plasma intercellular adhesion molecule (ICAM)-1 levels than C group at 0 and 6 h, whereas no difference in ICAM-1 concentrations were observed between the C and FO groups at 0 h after CLP. Hypercholesterolemia resulted in higher tissue MPO activities. There were no differences in MPO activities in various organs between the two experimental groups. These results suggest that hypercholesterolemic mice fed FO did not exhibit immunosuppression when complicated with sepsis. FO administration reduced adhesion molecule expressions and inflammatory-related mediators at the site of injury at an early but not a late stage of sepsis. However, compared with the SO group, the influences of FO on MPO activities in various organs were not obvious in hypercholesterolemic mice with sepsis.

Glutamine reduces the expression of leukocyte integrins leukocyte function-associated antigen-1 and macrophage antigen-1 in mice exposed to arsenic.

Chronic arsenic exposure results in an increased oxidative stress and inflammation in the body. Glutamine (GLN) is an amino acid considered to have immunomodulatory effects and attenuate the inflammatory reaction. This study was designed to examine the effect of GLN supplementation on inflammatory-related leukocyte integrin expression and in vitro splenocyte cytokine production in mice exposed to arsenic. Mice were assigned to the control and experimental groups. The control group drank deionized water, whereas the experimental group drank deionized water containing 50 ppm of sodium arsenite. Each control and experimental group was further divided into 2 subgroups and fed diets for 5 weeks. One subgroup was fed a semipurified diet, whereas the other subgroup was fed a diet where part of the casein was replaced with GLN, which provided 25% of the total amino acid nitrogen. The results showed that plasma GLN levels of mice in the arsenic group were significantly lower than those in the control groups. Glutamine supplementation reversed the depletion of plasma GLN in the arsenic group. beta(2) intergins, including leukocyte function-associated antigen-1 and macrophage antigen-1 expressed by leukocytes, were significantly higher in the arsenic group than the control groups. Glutamine supplementation reduced leukocyte integrin expression in mice exposed to arsenic. There were no differences in interleukin 4, interleukin 6, interferon gamma, and tumor necrosis factor alpha production between the 2 arsenic groups when splenocytes were stimulated with mitogen. These results suggest that arsenic exposure results in depletion of plasma GLN and higher leukocyte integrin expression. Glutamine supplementation normalized the plasma GLN levels and reduced leukocyte leukocyte function-associated antigen-1 and macrophage antigen-1 expression. However, cytokine modulation may not be responsible for reducing leukocyte integrin expression in mice exposed to arsenic.

Generation of cytotoxic T lymphocytes specific for B-cell acute lymphoblastic leukemia family-shared peptides derived from immunoglobulin heavy chain framework region.

BACKGROUND: Immunoglobulin heavy chain variable region (IgHV) is a well-characterized tumor antigen for B-cell malignancies. It can function as a target for T cell-mediated immune response. Clinical trials of IgHV protein vaccines against lymphoma have demonstrated induction of tumor-specific cytotoxic T lymphocyte (CTL) responses. However, complementary determining regions-based individual vaccines have disadvantages for wide clinical application. Although a recent study demonstrated that immunogenic peptides are derived from framework regions (FR) shared among patients with B-cell lymphoma, how to choose the appropriate peptides for each patient is still unsolved. The aim of this study was to investigate whether immunoglobulin heavy chain FR-derived peptides shared in each IgHV family are potential CTL epitopes presented by B-cell acute lymphoblastic leukemia (B-ALL). Such CTL epitopes might be beneficial to shifting vaccination strategies against B-ALL from individual specificity to family specificity. METHODS: Seven IgHV gene families were amplified respectively by PCR and sequenced directly from 71 childhood B-ALL cases. Bioinformatics was applied in analyzing characteristics of sequences available and predicting HLA-A*0201-restricted CTL epitopes for each IgHV family. An antigen-specific T cell expansion system was used to generate peptide-specific CTLs. The cytotoxicity of CTLs against B-ALL cells was assessed in the lactate dehydrogenase release assay. RESULTS: Complete IgHV rearrangements were identified in all of the 71 B-ALL cases. All of 40 sequences available showed > or = 98% homology with the nearest germline IgHV genes, indicating IgHV genes in B-ALL of germline nature. Twelve nonapeptides of high HLA-A*0201-binding scores were obtained from 26 productive IgHV protein sequences. Ten (83%) of the peptides were located in FR1 and FR3 shared among the corresponding IgHV family. CTLs specific for the peptide QLVQSGAEV located in FR1 (3 - 11) shared among the IgHV1 family could be successfully generated from peripheral blood mononuclear cells of two HLA-A*0201 + healthy donors in vitro and were capable of killing HLA-matched B-ALL cell clones belonging to the IgHV1 family. CONCLUSION: Anti-B-ALL CTLs against immunoglobulin heavy chain FR-derived peptides have family-specific cytotoxicity.

Effect of dietary fish oil supplementation on cellular adhesion molecule expression and tissue myeloperoxidase activity in diabetic mice with sepsis.

This study investigated the effect of n-3 fatty acids on adhesion molecules and tissue myeloperoxidase (MPO) activity in diabetic mice with sepsis. Diabetes was induced by a streptozotocin injection. Mice with blood glucose levels exceeding 2000 mg/l were considered diabetic. Diabetic mice were assigned to two groups with a medium-fat (10 %, w/w) diet either provided by soyabean oil (SO, n 30) or fish oil (FO, n 30). n-3 fatty acids provided 4.3 % of the total energy and the n-3/n-6 fatty acid ratio was 1:2 in the FO diet. After feeding the respective diet for 3 weeks, all mice had sepsis induced by caecal ligation and puncture (CLP) and were killed at 0, 6 or 24 h after CLP, with ten mice at each time-point. The result showed that compared with the SO group, FO group had lower PGE2 and TNF-alpha levels in peritoneal lavage fluid after CLP. Lymphocyte CD11a/CD18 expressions were higher at 6 h, whereas the percentage was lower at 24 h in the SO group than in the FO group. Neutrophil CD11b/CD18 expressions were significantly higher in the SO group than in the FO group at 0 h. The FO group had lower organ MPO activities at various time-points after CLP when compared with those of the SO group. The present findings suggest that compared with the diabetic mice fed SO, a low-dose n-3 fatty acid supplementation may attenuate leucocyte adhesion and infiltration into tissues in diabetic mice complicated with sepsis.

[Relationships between thiopurine methyltransferase gene polymorphisms and its enzymatic activity].

OBJECTIVE: To investigate the relationship between the thiopurine methytransferase (TPMT) gene polymorphisms and its enzymatic activity, and to clarify the significance of TPMT activity and gene polymorphisms on individualized therapy with thiopurines. METHODS: The TPMT activity and gene polymorphisms were determined in an unrelated population of 250 Chinese healthy blood donors, 100 cords blood and 280 patients with acute leukemia. The TPMT genotyping assay was based on polymerase chain reaction (PCR), restriction digestion of PCR products, denaturing high-performance liquid chromatography (DHPLC) and SNaPshot sequencing and direct DNA sequencing in the TPMT exon 5 (G238C), TPMT exon7 (G460A) and TPMTexon10 (A719G). Erythrocyte TPMT activity was measured by high-performance liquid chromatography (HPLC). RESULTS: The frequency of TPMT polymorphism in 250 Chinese healthy blood donors, 100 cords blood and 280 patients with acute leukemia was low (3.5%), and all the varied alleles were TPMT* 3C (exon 10A719G). All of them were TPMT* 1/TPMT* 3C heterozygote. The TPMT activity was between 6 and 12 U. The activity in 95.1% was more than 12 U (13 - 32 U), while the activity in others (4.9%) was 6 - 12 U. TPMT activity and genotype were concordant. Of 630 subjects evaluated, TPMT activity of heterozygous individuals in Chinese healthy blood donors, cords blood and acute leukemia patients were 9.1 U, 9.3 U and 9.07 U, respectively, significantly lower than that in general population (17.6 U, 17.67 U and 18.6 U, respectively). In the samples analyzed, ten subjects with heterozygous phenotypes (6/15 acute leukemia children and 4/16 healthy blood donors and cords blood) did not have TPMT* 2, TPMT* 3A or TPMT* 3C. Therefore, other factors may affect on TPMT activity. CONCLUSION: TPMT gene polymorphisms and its activity were concordant. The heterozygotes had low TPMT activity. Therefore, detection of TPMT genotype and its activity is useful. These findings hold a promise of improving the safety and efficacy of thiopurines therapy.

[Variant fusion transcript in ALL children with E2A-PBX1 fusion gene positive].

The study was aimed to investigate the expression of E2A-PBX1 fusion gene in children with acute lymphoblastic leukemia (ALL). The primers located at different sites of E2A and PBX1 gene were used to screen for the fusion gene in 410 children with ALL, including 362 cases of B cell ALL and 48 cases of T cell ALL. The results showed that 17 children carried the fusion gene. The positive rate was 4.1%. Furthermore, all the positive cases expressed a variant type of fusion transcript. It resulted from different splicing of the 13th exon (159 bp) of E2A gene. Analyses with BLASTn indicated that the variant type of transcript retained the open reading frame. However, the loss of 53 amino acid residues which were located at the 2nd activation domain resulted in the partial deletion of the putative loop-helix (LH) structure as well as the complete deletion of the heptad leucine repeat. It is concluded that all the children with ALL positive for the E2A-PBX1 fusion gene express typical and variant fusion transcripts. The latter resulted from different splicing of the 13th exon (159 bp) of E2A gene. The loss of 53aa would lead to the partial deletion of the putative loop-helix (LH) structure as well as the complete deletion of the heptad leucine repeat.

[Induction of anti-B-cell acute lymphoblastic leukemia cytotoxic T lymphocyte response against immunoglobulin heavy chain frame-derived nonapeptide].

OBJECTIVE: To induce anti-B-cell acute lymphoblastic leukemia (B-ALL) cytotoxic T lymphocyte response against immunoglobulin heavy chain frame-derived nonapeptide. METHODS: The peptide, QLVQSGAEV, containing IgHV1 frame region 3rd-11th amino acids (IgHV1(3-11)), was synthesized. IgHV1(3-11)-T2 binding tests were performed. HLA-A * 0201-positive normal peripheral blood mononuclear cells (PBMNC) were stimulated by IgHV1(3-11)-loaded antigen presenting cells three times at weekly intervals. HLA-A * 0201/IgHV1(3-11) tetramers were used to detect the proliferation of IgHV1(3-11)-specific CD8(+) T cells in the culture. Seven IgHV gene families of B-ALL patients were respectively amplified by PCR and the PCR products were sequenced to select IgHV1 and IgHV3 family monoallelic functional rearrangements. Among them, HLA-A * 0201 positive individuals were subsequently identified. Cytotoxicity of IgHV1(3-11)-specific CD8(+) T cells against HLA-A * 0201-positive IgHV1/IgHV3 family B-ALL cells was measured by MTT assay. RESULTS: The synthesized IgHV1(3-11) up-regulated HLA-A * 0201 expression on T2 cell surface by 1.63-folds. The percentage of IgHV1(3-11)-specific CD8(+) T cells in HLA-A * 0201-positive normal PBMNC was increased from 1.64% after second stimulation to 82.57% after third stimulation. At effector: target ratio of 20:1, the killing rate of IgHV1(3-11)-specific CD8(+) T cells against IgHV1 family B-ALL cells was 18.24%, being 1.8-folds as that against IgHV3 family B-ALL cells (P = 0.01). CONCLUSION: Cytolytic T lymphocytes generated in vitro against immunoglobulin heavy chain frame-derived nonapeptides can kill B-ALL cells expressing these peptides.

[Epitopes recognized by cytotoxic T lymphocytes in immunoglobulin heavy chain variable regions expressed by B-cell acute lymphoblastic leukemia].

OBJECTIVE: To clone IgHV genes from childhood B-ALL cells and identify CTL epitopes deduced from IgHV gene. METHODS: Seven IgHV gene families were respectively amplified by PCR and directly sequenced for 37 childhood B-ALL cases. Bioinformatics were applied for analyzing characteristics of sequences available and predicting HLA-A*0201 molecule-binding nonapeptides derived from IgHV. The predicted nonapeptide QLVQSGAEV was synthesized and its binding affinity to T2 cells determined. CD8+ T cells from a healthy HLA-A*0201+ donor peripheral blood were stimulated repeatedly with QLVQSGAEV-loaded antigen presenting cells. RESULTS: IgHV gene rearrangements were identified in 37 B-ALL. Forty IgHV gene sequences available preferentially utilized V(H)4-59 and V(H)4-34 gene segments. Increased frequency (15.4%) of D7-27 in B-ALL IgHV was found compared to that reported for adult PBLs (P = 0.02); 20.0% DJ(H) junctions in B-ALL lacked non-encoding nucleotides, a frequency higher than that reported for adult PBLs (P = 0.02). 17.5% B-ALL IgHV contained < 2% replacement mutations. Forty B-ALL IgHV sequences acquired 12 high HLA-A*0201-binding nonapeptides, 10 (83.0%) peptides were located in frame region (FR)1 and 3. The synthesized peptide QLVQSGAEV up-regulated HLA-A*0201 expression 1.63 fold on the surface of T2 cells. The frequency of QLVQSGAEV-specific CD8+ T cells in a healthy HLA-A*0201+ donor peripheral blood increased from 1.6% and 82.6% after two-round and 3-round stimulations, respectively. CONCLUSION: IgHV genes in childhood B-ALL are of germline characteristics. Their heavy chain framework regions contain HLA-A*0201-binding nonapeptides. These peptides are capable of inducing specific CD8+ T cells to activate and proliferate.

[Characteristics of immunoglobulin heavy chain variable region genes in childhood B-cell acute lymphoblastic leukemia].

OBJECTIVE: To investigate the origin of childhood B-cell acute lymphoblastic leukemia (B-ALL) and its epitopes recognized by cytotoxic T lymphocytes (CTL) in immunoglobulin heavy chain variable region (IgHV). METHODS: Seven IgHV gene families were respectively amplified by PCR and directly sequenced in 108 childhood ALL. The amino acid sequences were deducted from sequenced nucleotides. Bioinformatics was applied to analyses of recombination patterns, somatic mutations and germline gene segments usage, and to prediction of epitopes recognized by CTL. RESULTS: IgHV gene rearrangements were identified in 66% of the cases, including 37 (52.1%) monoallelic rearrangements, 26 (36.6%) biallelic rearrangements and 8 (11.3%) oligoclonal rearrangements. Among the obtained 40 B-ALL IgHV gene sequences, 8 (20.0%) were in frame rearrangements without stop codons. V(H3) (11/40), V(H4) (11/40) and V(H1) (8/40) amounted to 75% rearranged V(H) families. V(H)(4-59) and V(H)(4-34) were the most frequently rearranged V(H)(4) family gene segments. Usage of D2 and D3 families was most prominent (35.9% and 28.2%, respectively). Increased frequency of D7-27 (15.4%) was found as compared to that of normal peripheral B lymphocytes (P = 0.02). J(H)(6) was found in 47.5% rearrangements followed by J(H)(4) (27.5%). 8/40 (20.0%) DJ(H) junctions lacked N nucleotides, which was higher than that reported for normal peripheral B lymphocytes (P = 0.02). 17.5% B-ALL IgHV contained scattered replacement mutations with replacement (R) to silent (S) substitution ratio (R/S ratio)

[Analysis of T cell repertoire in children with acute B lymphoblastic leukemia].

OBJECTIVE: The complementarity determining region 3 (CDR3) of T cell receptor (TCR) is the place through which T cells connect to the antigen. The lengths and DNA sequences of CDR3s are different according to different T cell clones. This leads to a diverse TCR CDR3 repertoires which can reflects the functional status of T cells precisely. This study aimed at elucidating the abnormality of TCR beta chain variable region (BV) CDR3 repertoires of children with acute B lymphoblastic leukemia, the pathogenesis of leukemia associated with T cell dysfunction and the immuno-reconstruction of T cells after the chemotherapy. METHODS: Twelve children aged from 3 to 13 years (average 4.50 +/- 3.78 years) with acute B lymphoblastic leukemia before chemotherapy and 8 healthy control donors aged from 6 to 16 years (average 10.30 +/- 3.00 years) were enrolled. Four of 12 patients were studied for the second time 3 months after complete remission (CR). Reverse transcription-polymerase chain reaction (RT-PCR) and polyacrylamide sequencing gel electrophoresis were used to detect the diversity of TCR BV CDR3 repertoires of these children. RESULTS: (1) The expression of BV2 and BV3 in 12 children increased and BV17 and BV18 decreased before the chemotherapy as compared with controls (P < 0.05). There were 4 children with a lower level expression of BV21 before the chemotherapy, and much lower level expression was found after the remission. (2) Normally each lane contained eight to ten bands, which represented unique CDR3 sizes for a given TCR BV family. Bands differed in size by 3 nucleotides and generally form a Gaussian distribution. There were 14% TCR BV families with abnormal CDR3 length distribution in 12 patients before therapy, which was significantly higher than that of controls (5.5%, P < 0.05). Restricted CDR3 length distribution was observed in BV14, BV1, BV16, BV20, BV13.1, BV13.2 and BV6. Every abnormal BV family recovered to normal Gaussian distribution 3 months after CR. CONCLUSION: T lymphocytes in children with acute B lymphoblastic leukemia revealed markedly skewed repertoires, which suggests the abnormality of T cell functions. Most abnormal T cell repertoires recovered to normal Gaussian distribution 3 months after the first CR, which suggests the immunological reconstitution of T cell repertoires.

[Proliferation of specific cytotoxic T lymphocytes induced by immunoglobulin heavy chain framework region-derived antigenic nonapeptides].

OBJECTIVE: To identify that immunoglobulin heavy chain framework regions-derived peptides function as cytotoxic T lymphocytes epitopes. METHODS: Seven IgHV gene families were respectively amplified by PCR and directly sequenced for 108 acute lymphoblastic leukemia cases. Sequences available were translated into amino acid sequences. Bioinformatics were applied for analyzing recombination patterns and gene mutations of the IgHV genes. Softwares SYFPEITHI and BIMAS were used for predicting the T cell epitopes in the immunoglobulin heavy chain variable regions. To determine whether the predicted peptides have immunogenicity, a IgHV1 family nonapeptide QLVQSGAEV was synthesized as a representation and T2 binding assay of this peptide was performed, inducing proliferation of T cells from normal HLA-A * 0201 peripheral blood lymphocytes (PBLs) with QLVQSGAEV-loaded antigen presenting cells, and detecting the proliferating T cells by HLA-A * 0201/QLVQSGAEV tetramers. RESULTS: Complete IgHV gene rearrangements were identified in 66% cases. Among 40 B-ALL IgHV sequences available, 26 were predicted for antigenic nonapeptides that are likely to bind to HLA-A * 0201 molecule. Twelve peptides were acquired. Except one peptide derived from CDR3, 10 (83%) peptides were located in the immunoglobulin heavy chain framework regions. Moreover B-ALL belonging to the same IgHV family shared 1 - 2 peptides. Synthesized peptide QLVQSGAEV up-regulated HLA-A * 0201 expression 1.63 times on T2 cell surface. PBLs from a normal HLA-A * 0201 donor were stimulated with QLVQSGAEV-loaded autologous PBMCs and T2, the CD8(+) tetramer(+) cells in gated lymphocyte population increased from 1.64% after the first stimulation to 82.57% after the third stimulation. CONCLUSION: Immunoglobulin heavy chain framework region genes encode IgHV family-specific peptides recognized by CTLs. Specific CTLs remain in human peripheral T cell repertoire. Immunoglobulin heavy chain framework-derived peptides function as T cell epitopes to induce the proliferation of specific CTLs.